Particle Sensor with Interferent Discrimination

ABSTRACT

This invention provides methods and devices to measure particle suspension concentrations in the presence of potential interferents. Particle back-scatter readings are taken at light wavelengths that are absorbed by the medium before interacting with surrounding objects. Source-detector spacings are minimized compared to the mean absorbance path length of light, thereby maximizing the range of sensitivity to particle concentration. Discrimination against potentially interfering particles, such as bubbles, is provided by mapping the signal distribution against the central signal value and/or by the use of statistical measures with reduced dependence on outliers. The methods and devices allow accurate particle concentration readings over a wide range of concentration in environments crowded with potentially interfering objects and in the presence of variable concentrations and sizes of potentially interfering particles.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application from U.S. patentapplication Ser. No. 15/679,465, which is a Divisional application fromparent application Ser. No. 14/691,421, which is related to, and claimsbenefit of U.S. Provisional Patent Application Ser. No. 61/982,087 filedApr. 21, 2014, the disclosure of which is incorporated herein byreference in its entirety for all purposes.

FIELD OF THE INVENTION

Methods and devices for accurately measuring particulate concentrationsin a suspension, e.g. biomass in a liquid cell culture, that may includemoving interfering objects, such as bubbles, and stationary interferingobjects, such as immersed sensors. Measurements of particleconcentration are made, e.g., by detection of near infrared (NIR)back-scattered light. Detected signals can be related to, e.g., particleconcentration, biomass, or standard O.D. 600 nm values.

BACKGROUND OF THE INVENTION

Measurement of particle concentration is important in many industrialand research applications. For example, monitoring cell density (e.g.biomass) in liquid cell cultures is used: to determine the growth phaseor rate; as a feedback signal for adjusting growth conditions (e.g.dissolved oxygen, pH, media constituents); and/or as an indicator ofwhen to induce expression of genes, harvest cells, or inoculate cellsinto a larger media volume. The growth rate of many cultures,particularly microbial organisms (e.g. yeast, bacteria), is limited bythe concentration of dissolved oxygen in the medium. The culture of suchorganisms is often performed in vessels (e.g. fermenters, bioreactors)in which gases are bubbled (“sparged”) and the medium is stirred orotherwise agitated, often at such high rates that the bubbles constitutea significant fraction (“gas hold-up”) of the total volume. The presenceof such a high concentration of bubbles presents a challenge to manytechniques for cell growth monitoring.

Many biomass monitoring techniques take advantage of the scattering oflight by cells. For example, one of the most common laboratorytechniques for monitoring cell growth is to extract a sample, dilute it,and measure it's absorbance (e.g. at 600 nm) in a fixed path length(e.g. 1 cm) cell in a spectrophotometer. Absorbance is typically limitedto about 0.5 in order to remain in the linear range of Beer's Law. Themeasured absorbance multiplied by the dilution factor is referred to asthe optical density (e.g. “OD(600nm)”), and used as an indication ofbiomass. Despite its prevalence, this technique has numerouslimitations: it requires opening the culture, with the attendant risk ofcontamination; the dilution step is subject to volumetric error; theextracted sample is expended, of particular concern in small volumecultures; and it is labor-intensive.

In an effort to overcome these limitations and provide continuous(“on-line”) monitoring, much work has gone into the development ofinvasive sensors for measuring optical density directly in the cellculture. Unfortunately, such sensors are subject to the same limitationof narrow linear range as are off-line techniques: in order to measurebiomass over a wide range, the use of multiple sensors, having differentoptical transmission path lengths, is frequently required. Immersiblesensors that measure back-reflected light (instead of transmission)typically have a somewhat wider but still limited linear range ofresponse to biomass, and may suffer, particularly in the low biomassrange, from a sensitivity to reflections from nearby non-biologicalobjects within the vessel, such as impellers, pH sensors, etc. This canrender the sensors inaccurate, in an unpredictable way.

Methods for monitoring biomass non-invasively, through the vessel wall,or through an optical window, have been developed in recent years. InU.S. Pat. No. 7,100,462 “Self Adjusting Sensor Mounting Device”, methodsand devices are described for reproducibly mounting a sensor to a widevariety of cylindrical and flat surfaces in a manner that automaticallycompensates for the curvature of the mounting surface.

In U.S. Pat. No. 8,603,772, “Particle sensor with wide linear range”,methods and devices are described for measuring particulateconcentration in vessels, where the response from multiplesource-detector pairs is combined to provide a linear response over awide range of particle concentrations. Also described, are methods anddevices for confining the measurement to a specific volume within themedium, as methods and devices for performing rapid sequentialmeasurement of particle concentration in multiple vessels.

In U.S. Pat. No. 8,405,033 “Optical sensor for rapid determination ofparticulate concentration”, methods and devices are described forlimiting the optical penetration depth of measurements of particledensity by the use of light at wavelengths that are strongly absorbed bythe medium, and matching the source-detector separation to theabsorbance path length.

In view of the above, a need exists for devices that can readparticulate concentrations accurately in the presence of bubbles as wellas other nearby reflective objects, that is not prone to fouling, andthat is linear over a wide range of biomass. Benefits could also berealized from methods and devices capable of reading particleconcentrations in shallow samples and without the need to dilute thesample, or employ multiple sensors. The present invention provides theseand other features that will be apparent upon review of the following.

SUMMARY OF THE INVENTION

An optical sensor for accurately measuring the concentration ofparticles suspended in a medium while mitigating interference from otherobjects in the medium is disclosed. The sensor consists of one or morelight sources and one or more detectors contained in a housing. Thelight sources and detectors are selected to emit and detect light in aspectral region that is substantially absorbed by the medium, therebyconfining the measurement to a restricted volume within the medium. Insome embodiments statistical measures of the central value anddistribution of the detected light signal are combined to providediscrimination between particle types. Additionally described aremethods and devices for accurately determining the particleconcentration over a wide range of particle concentrations.

Methods of the invention can include selecting the source wavelength tohave a mean absorption path length in the medium that is substantiallygreater than the separation between the central optical axes of thelight source and detector at the interface with the medium, while at thesame time being substantially smaller than the distance between theoptical interface with the medium and the nearest potentiallyinterfering stationary object falling within the emission cone of thelight source.

In specific embodiments in which the medium is aqueous and the suspendedparticle to be measured is biological (i.e. cells, micro-organisms,etc.), selection of source wavelength(s) having sufficient absorbance bywater so that the emitted light is substantially attenuated beforeinteracting with non-biological objects in the container prevents theseobjects from influencing measurement of the biological particles. Byalso selecting the source-detector separation to be substantially lessthan the mean absorption path length, a wide range of sensitivity tochanges in biological particle concentration is allowed.

In certain embodiments the medium includes more than one type ofparticle, and discrimination between the particle types is enabled. Insome such embodiments the detection bandwidth and measurement volume arerestricted so that signal fluctuations due to a second particle type ofparticle are observed, whereas the signal due to a first particle typeis substantially constant at a given concentration. In some embodiments,the medium is aqueous and the first particle type is a biologicalparticle and the second particle type is a gas bubble. The measurementvolume and detection bandwidth are selected so that the fluctuations dueto gas bubbles are detected as the number or size of the bubbles withinthe measurement field varies. The biological particles within the samemeasurement volume may be substantially smaller and more numerous thanthe gas bubbles, so that the detected fluctuations due to the biologicalparticles is substantially less than that of the bubbles.

In some embodiments the detected signals as characterized according totheir central value. In further embodiments, the method for determiningthe central value has reduced sensitivity to outliers compared to themean. Examples of such methods are the median, mode, and trimmed mean.Such methods may provide the ability to discriminate between differentparticle types, particularly in situations where the particle types havedifferent signal distributions. In other embodiments, the detectedsignals are further characterized according to their distribution. Thecentral value and distribution estimates may be combined to determinethe concentration of a particle type, while being substantiallyinsensitive to the concentration of another particle type.

The methods can further include a means of detecting the position ororientation of the sensor relative to the container, or providing meansof reducing the sensitivity of the measurement result to variations inthe thickness of the window. The method can include providing means ofdetecting the position and orientation of the sensor relative to thecontainer. For example, one or more position switches can provide meansto determine the position or orientation of the sensor relative to thecontainer. Optionally, one or more capacitance switches can provide ameans to determine the position or orientation of the sensor relative tothe container. For example, the capacitance switches can be used todetermine that a sufficient volume of medium is present in the vicinityof the sensor for a particle concentration measurement to be madeaccurately. Alternately, the position or orientation can be determinedusing optical components, such as a light source and detector.

In certain embodiments, radiation sources and sensors are positionedsymmetrically about their counterpart. For example, at least two sourcescan be positioned symmetrically with respect to a detector component, orvice versa. Such devices can reduce inconsistent detections or detectpositioning errors during analysis of a sample. For example, the twosymmetrically positioned optical elements can provide a means ofdetermining whether sufficient medium is present in order to make anaccurate measurement of particle concentration. Comparison of the two ormore measurements may also provide a means of detecting obstructions orinterferences at or near the optical interface. For example a stationarybubble or debris in the vicinity of the optical elements may havedifferent effect on the measurements at different locations. Suchcomparison may be useful for excluding or reducing the influence ofmeasurements made under conditions where such interferents are present.

In preferred embodiments, the radiation is detected as back-scatteredlight and the result is converted to an optical density readout. In manyembodiments, absorbing components of the medium being analyzed includewater.

In many embodiments of the methods, a means is provided for subtractinga measurement of blank media from a measurement of media containingparticulate matter. In preferred embodiments, coefficients can be inputor determined relating detected signals to the particle concentrationmeasurement. In the methods, it is beneficial to provide a means ofchecking for or correcting for proper instrument performance, e.g., byproviding duplicate measurements, measurements from different lightpaths, comparison to standard references, comparison to controls, etc.

In certain methods, a guide is provided for guiding the sensor into adesired alignment with the container. In preferred embodiments, theguide includes a U or V-shaped feature in one dimension and asubstantially flat feature in a second dimension, e.g., to align thesensor relative to the axis of a sample container. In some embodiments,spring force is used to hold the guide against the sensor. In manyembodiments, the guide is located on the opposite side of the containerfrom the sensor, so that multiple different container sizes may beautomatically accommodated.

In one embodiment, the sensor consists of at least one near-infraredlaser light source emitting near 1550 nm, and at least one opticalreflectance detector. The sensor is held against the wall of a vessel,such as a test tube, containing cells or microorganisms suspended in anaqueous medium. Laser light is directed through the wall of the vesselinto the medium and is scattered by the cells or microorganisms. Inaddition to scattering, the 1550 nm light is partially absorbed by waterin the medium. The relationship between Optical Density and thereflectance signal measured by the sensor is stored as a calibration inthe instrument, which may vary according to the type of cell ormicroorganism being grown in the medium. In some embodiments, a baselineoffset is provided to allow compensation for the contribution of themedium to the measured Optical Density.

In preferred embodiments, the radiation source emits light at afrequency ranging from about 650 nm to 2200 nm, from 700 nm to 2000 nm,from 800 nm to 1800 nm, from 1000 nm to 1600 nm, or about 1500 nm. Inpreferred embodiments, the source provides light radiation in theinfrared wavelengths. In some embodiments the source provides radiationranging between 1150 nm and 1350 nm. In other embodiments, the sourceemits light at a wavelength between 920 and 1150 nm, or between 350 and1900 nm.

In some embodiments a second reflectance or scatter detector is arrangedsymmetrically with respect to a first reflectance detector, as a meansto determine proper sensor positioning. Comparison of the processedsignals from the two detectors ensures that the measurement is beingmade from within a sufficient depth of fluid to provide accuratedetermination of particle concentration, and that interfering objectsare not present at or near interface between the sensor and container.In some embodiments additional detectors are spaced at a differentdistance from the laser to extend the linear range of response tochanges in particle concentration.

In some embodiments the reflectance detectors are held at the end ofapertures constructed from a material, which is strongly absorptive oflight emitted by the source. The optic axes of the laser and reflectancedetectors may be oriented parallel to each other, and the divergence andcollection angles restricted, so as to minimize sensitivity to vesselwall thickness and restrict the measurement volume within the medium.

In some embodiments, the sensor includes at least one position detector.The position detector(s) on the sensor face provide a means ofdetermining when proper sensor positioning has been achieved, and canalso be used to trigger a measurement and to help determine whether ameasurement has been successfully completed. In some embodiments theposition sensor includes at least one position switch. In otherembodiments the position sensor includes at least one capacitancesensor. In some embodiments the capacitance sensor is used to determinewhether sufficient medium is present in the vessel in proximity to thesensor in order for an accurate measurement to be collected. In otherembodiments, the position sensing is performed using optical components.

In some embodiments the concentration is reported as an Optical Density(OD) such as would be reported by a spectrophotometer at a particularwavelength (e.g., 600 nm) through a 1 cm path length cell containing adilute solution of the medium, after multiplication by the dilutionfactor. In some embodiments the concentration is reported as standardturbidity units (e.g., NFUs), dry or wet weight per volume (e.g., g/mL),cells counts per volume (e.g., cell/mL), or any user-defined value,related to particle concentration. In some embodiments the type of unitsin which the result is reported is user-selectable.

The light scatter by particles in the methods can be detected by anyappropriate type of detector. For example, detection can be by aphotomultiplier tube, photodiode, or photodiode array. In preferredembodiments, at least one of the detectors employs silicon in activedetector area of its sensor. In some embodiments, at least one of thedetectors employs InGaAs in its active area.

The illumination source in the methods can be any appropriate means,such as, e.g., a laser, a tungsten lamp, mercury vapor lamp, LED, diodelasers and/or the like. In certain embodiments, a monitoring diode isbuilt into the illumination source, and is used to compensate forchanges in radiant flux emitted by the source, e.g., a laser monitoringdetector is used to directly measure the output of the laser, providinga means of compensating for intensity variation such as may be caused bytemperature fluctuations.

In some methods of the invention, a bar code scanner is built into thedevice and used to track the measurements, e.g., the identification ofsamples. In the methods, it can be useful to have informationtransferred wirelessly, e.g., the measured particle concentration can bewirelessly transferred to an ancillary or peripheral device.

In another embodiment of the methods, a concentration of at least onetype of particle in a medium is determined by positioning a sensor nextto a container holding the medium, passing radiation originating from atleast one source through the container wall into the medium thatsubstantially absorbs the radiation, detecting with at least onedetector a signal relating to radiation reflected from within saidmedium back through the container wall, and relating the detectedradiation signals to the concentration of a particle in the medium.

The inventions include devices for detecting and measuring theconcentration of particles in a container. In one aspect, the device fordetermining the concentration of at least one type of particle in amedium includes, e.g., a housing containing a sensor, including at leastone radiation source and at least one radiation detector positioned tocollect source radiation scattered by particles within said medium,wherein the radiation emitted by the source is substantially absorbed bythe medium. The device further comprises a controller for controllingthe radiation sources, and for measuring at least one of the signalscorresponding to the portion of radiation detected by one of thedetectors that originated from the radiation sources. The devicetypically also includes a processor configured to relate at least one ofthe signals to a concentration of at least one type of particle.

The device sensor can be configured to be well-adapted to takingmeasurements from any number of different containers or vessels. Forexample, the sensor housing can be attached to a disposable fermentor,bioreactor, flask, bottle, bag, or tube. The sensor housing can beaffixed to a vessel, providing the capability of making multiplemeasurements without the need for reapplication of the sensor to thevessel. Multiple sensors can be interfaced with the same controller. Insome embodiments, the sensor housing is designed to be disposable.Radiation sources or detectors can be fiber optical components opticallylinked to electro-optical components that are physically separated fromsaid housing. In some embodiments fiber optics are used to convey lightbetween the sensor and electro-optical components. In some suchembodiments fiber optics splitters and/or switchers are used tomultiplex the electro-optical components between multiple fibers.

The device can be configured to take particle concentrationmeasurements. In preferred embodiments, the time required formeasurement of particle concentration is three seconds or less. In otherembodiments, the measurement time is variable, as determined by a metricrelated to measurement accuracy. In another aspect, signals are combinedfrom source-detector pairs with at least two different separationdistances. In another aspect, at least two of the radiation sources havesubstantially different emission wavelengths. In certain devices, thesource wavelengths can be selected according to the separation betweenthe source-detector pairs.

In some embodiments the sensor is immersed in the medium. In some suchembodiments the materials comprising the sensor face include glass. Insome such embodiments the glass is curved so as to discourage theaccumulation of objects on the exposed surface. In embodiments where thesensor is immersed in a liquid medium, the use of a glass face,particularly with a curved shape, discourages the accumulation of gasbubbles or debris on the exposed surface. In other embodiments thematerials comprising the sensor face include stainless steel that hasbeen coated with a material, such as titanium or zirconium nitride, thatdiscourages the accumulation of bubbles or biological material on thesensor surface.

In some embodiments fiber optics are used to aid in the transport oflight from the light source(s) and to the detector(s). This may allowthe optical components to be positioned remotely from the point ofoptical interface with the medium.

Definitions

Unless otherwise defined herein or below in the remainder of thespecification, all technical and scientific terms used herein havemeanings commonly understood by those of ordinary skill in the art towhich the present invention belongs.

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular devices orbiological systems, which can, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting. As used in this specification and the appended claims, thesingular forms “a”, “an” and “the” include plural referents unless thecontent clearly dictates otherwise. Thus, for example, reference to “acomponent” can include a combination of two or more components;reference to “feed” can include mixtures of feed, and the like.

Although many methods and materials similar, modified, or equivalent tothose described herein can be used in the practice of the presentinvention without undue experimentation, the preferred materials andmethods are described herein. In describing and claiming the presentinvention, the following terminology will be used in accordance with thedefinitions set out below.

The sensors described herein include some embodiments in which the lightsource and detector are physically located within the sensor(“free-space embodiments”), and other embodiments in which the lightsource and detector are located outside of the sensor, and light isconveyed to and from the sensor through the use of fiber optics, whichmay include single fibers or bundles of fibers (“fiber-opticembodiments”). As used herein, when reference is made to a “lightsource” or “detector” in a sensor, in addition to free-space embodimentsthe meaning also encompasses fiber optic embodiments, where the fiberoptics act as an extension of the light source and/or detector.

As used herein, an “absorption coefficient” of a medium is the negativeof the logarithm of the ratio of light transmitted through the medium tothe light transmitted in the absence of the medium, divided by the pathlength travelled through the medium.

As used herein, an “detection cone” of a detector is the cone ofdetection rays surrounding the central optical axis of the detector atwhich the detected intensity is half that detected at the centraloptical axis.

As used herein, an “emission cone” of a light source is the cone ofemission rays surrounding the central optical axis of the light sourceat which the light source intensity is half that at that the centraloptical axis.

As used herein, a “mean absorption path length” in a medium is theinverse of the absorption coefficient of the medium.

A “sensor” of the methods and devices is a device component comprisingat least one light source-detector pair in functional association.

As used herein, “modulated” means to vary the amplitude, frequency, orphase of a light source.

As used herein, “substantially” refers to largely or predominantly, butnot necessarily entirely, that which is specified.

The term “about”, as used herein, indicates the value of a givenquantity can include quantities ranging within 25% of the stated value,or optionally within 10% of the value, or in some embodiments within 1%of the value.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a depicts an end view of a fiber optic sensor used to demonstratecertain aspects of the present invention.

FIG. 1b depicts an end view of the same fiber optic sensor as in FIG. 1a, but with a different configuration of connections to sources anddetectors.

FIG. 2 is a graph of optical penetration depth as a function of yeastconcentration for several configurations of source-detector pairs of thesensor depicted in FIG. 1 a.

FIG. 3 is a graph of median signal as a function of yeast concentrationmeasured by a sensor of the present invention on a bioreactor.

FIG. 4a is graph of back-scattering amplitude as a function of time fora fiber optic source-detector pair immersed in fermentor containing 25g/L yeast, without bubbling. The source and detector fibers are S1 andD1, as depicted in FIG. 1 b.

FIG. 4b is a histogram of the data graphed in FIG. 4 a.

FIG. 4c is graph of back-scattering amplitude as a function of time fora fiber optic source-detector pair immersed in fermentor containing 25g/L yeast, with vigorous bubbling. The source and detector fibers are S1and D1, as depicted in FIG. 1 b.

FIG. 4d is a histogram of the data graphed in FIG. 4 c.

FIG. 4e is graph of back-scattering amplitude as a function of time fora fiber optic source-detector pair immersed in fermentor containing 0.4g/L yeast, with no bubbling. The source and detector fibers are S1 andD1, as depicted in FIG. 1 b.

FIG. 4f is a histogram of the data graphed in FIG. 4 e.

FIG. 4g is graph of back-scattering amplitude as a function of time witha fiber optic source-detector pair immersed in fermentor containing 0.4g/L yeast, with vigorous bubbling. The source and detector fibers are S1and D1, as depicted in FIG. 1 b.

FIG. 4h is a histogram of the data graphed in FIG. 4 g.

FIG. 5a is a graph of standard deviation as a function of mean valuemeasured with a sensor of the present invention having a sourcewavelength of 1310 nm, and a source-detector separation of about 7 mm(S1-D4 in FIG. 1b ), that was immersed in a 12L bioreactor containingvarious concentrations of yeast dissolved in 10L of 0.9% saline, andagitated and sparged with air over a wide range of settings (400-900rpm, 0-16 lpm, respectively). Measurements made at the same yeastconcentration (but differing sparge and agitation) are represented withmarkers having the same shape and color.

FIG. 5b is a graph of standard deviation as a function of mean valuemeasured with a sensor of the present invention having a sourcewavelength of 1310 nm, and a source-detector separation of about 3.4 mm(S1-D3 in FIG. 1b ), that was immersed in a 12L bioreactor containingvarious concentrations of yeast dissolved in 10L of 0.9% saline, andagitated and sparged with air over a wide range of settings (400-900rpm, 0-16 lpm, respectively). Measurements made at the same yeastconcentration (but differing sparge and agitation) are represented withmarkers having the same shape and color.

FIG. 5c is a graph of standard deviation as a function of mean valuemeasured with a sensor of the present invention having a sourcewavelength of 1310 nm, and a source-detector separation of about 1.2 mm(S1-D2 in FIG. 1b ), that was immersed in a 12L bioreactor containingvarious concentrations of yeast dissolved in 10L of 0.9% saline, andagitated and sparged with air over a wide range of settings (400-900rpm, 0-16 lpm, respectively). Measurements made at the same yeastconcentration (but differing sparge and agitation) are represented withmarkers having the same shape and color.

FIG. 5d is a graph of standard deviation as a function of mean valuemeasured with a sensor of the present invention having a sourcewavelength of 1310 nm, and a source-detector separation of about 0.22 mm(S1-D1 in FIG. 1b ), that was immersed in a 12L bioreactor containingvarious concentrations of yeast dissolved in 10L of 0.9% saline, andagitated and sparged with air over a wide range of settings (400-900rpm, 0-16 lpm, respectively). Measurements made at the same yeastconcentration (but differing sparge and agitation) are represented withmarkers having the same shape and color.

FIG. 5e is a graph of median absolute deviation as a function of medianvalue measured with a sensor of the present invention having a sourcewavelength of 1310 nm, and a source-detector separation of about 0.22mm, that was immersed in a 12L bioreactor containing variousconcentrations of yeast dissolved in 10L of 0.9% saline, and agitatedand sparged with air over a wide range of settings (400-900 rpm, 0-16lpm, respectively). Measurements made at the same yeast concentration(but differing sparge and agitation) are represented with markers havingthe same shape and color.

FIG. 5f is a graph of median absolute deviation as a function of medianvalue measured with a sensor of the present invention having a sourcewavelength of 1550 nm, and a source-detector separation of about 0.22 mm(S2-D1 in FIG. 1b ), that was immersed in a 12L bioreactor containingvarious concentrations of yeast dissolved in 10L of 0.9% saline, andagitated and sparged with air over a wide range of settings (400-900rpm, 0-16 lpm, respectively). Measurements made at the same yeastconcentration (but differing sparge and agitation) are represented withmarkers having the same shape and color.

FIG. 6a is a power spectrum graph of the logarithm of the reflectancesignal as a function of sampling frequency measured with a sensor of thepresent invention having a source wavelength of 1310 nm, and asource-detector separation of about 0.22 mm (S1-D1 in FIG. 1b ), thatwas immersed in a 12L bioreactor containing 200 g/L yeast dissolved in10L of 0.9% saline, agitated at 900 rpm and sparged with air at 16 lpm.

FIG. 6b is a power spectrum graph of the logarithm of the reflectancesignal as a function of sampling frequency measured with a sensor of thepresent invention having a source wavelength of 1310 nm, and asource-detector separation of about 0.22 mm (S1-D1 in FIG. 1b ), thatwas immersed in a 12 L bioreactor containing 6 g/L yeast dissolved in 10L of 0.9% saline, agitated at 900 rpm and sparged with air at 16 lpm.

FIG. 6c is a power spectrum graph of the logarithm of the reflectancesignal as a function of sampling frequency measured with a sensor of thepresent invention having a source wavelength of 1310 nm, and asource-detector separation of about 0.2 mm (S1-D1 in FIG. 1b ), that wasimmersed in a 12L bioreactor containing 6 g/L yeast dissolved in 10L of0.9% saline, agitated at 400 rpm and with no air sparging.

FIG. 7a is a graph of the same data as in FIG. 5e after decimating thedata by a factor of two, and then truncating the data by a factor offour.

FIG. 7b is a graph of the same data as in FIG. 5f after decimating thedata by a factor of two, and then truncating the data by a factor offour.

FIG. 8 is a model of the source-detector overlap through a containerwall and into a medium for a sensor of the present invention.

FIG. 9 is an example of a timing scheme for laser driving and detectorreading for a device of the present invention.

FIG. 10a depicts an embodiment of a free-space optical arrangement of asensor of the present invention employing one source and one detector.

FIG. 10b depicts an embodiment of a free-space optical arrangement of asensor of the present invention employing two sources and one detector.

FIG. 10c depicts an embodiment of a free-space optical arrangement of asensor of the present invention employing one source, one detector, anda polarizing beam splitter.

FIG. 10d depicts an embodiment of a free-space optical arrangement of asensor of the present invention employing two sources, one detector, anda polarizing beam splitter.

FIG. 11a depicts an embodiment of a sensor of the present inventionemploying one fiber optic for both light delivery and collection.

FIG. 11b depicts an embodiment of a sensor of the present inventionemploying one fiber optic for light delivery and a second fiber opticfor light collection.

FIG. 11c depicts an embodiment of a sensor of the present inventionemploying separate fiber optics for light delivery and collection, and athird fiber that can be configured for either light delivery or lightcollection.

FIG. 11d depicts an embodiment of a sensor of the present inventionemploying separate fiber optics for light delivery and collection, andfive additional fibers that can be configured for either light deliveryor light collection.

FIG. 11e depicts an embodiment of a sensor of the present inventionemploying separate fiber optics for light delivery and collection, and athird fiber in a triangular arrangement that can be configured foreither light delivery or light collection.

FIG. 12a is a side view of an embodiment of a sensor of the presentinvention which includes a flat surface on the sensor face and ascattering reference material held in the closed position.

FIG. 12b is a side view of an embodiment of a sensor of the presentinvention which includes a rounded surface on the sensor face and ascattering reference material held in the closed position.

FIG. 12c is a side view of an embodiment of a sensor of the presentinvention which includes a rounded surface on the sensor face and ascattering reference material held in the open position.

FIG. 13a is an isometric view of an embodiment of a sensor of thepresent invention designed for measurements through the wall of a testtube.

FIG. 13b is a second isometric view of an embodiment of a sensor of thepresent invention designed for measurements through the wall of a testtube (the test tube is removed in this view).

FIG. 13c is a top view of an embodiment of a sensor of the presentinvention designed for measurements through the wall of a test tube.

FIG. 14 is an embodiment of a sensor of the present invention designedfor measurements through the wall of a small disposable plastic vessel.

FIG. 15 is an example of a bubble calibration of the present inventioncollected with a sensor of the design depicted in FIG. 14.

FIG. 16 summarizes results of applying the bubble calibration shown inFIG. 15 to new measurements.

FIG. 17 provides an example of the linear relationship between biomassand bubble-compensated reflectance according to an embodiment of thepresent invention.

FIG. 18 depicts an embodiment of a probe of the present inventiondesigned for immersion into small bioreactor vessels.

FIG. 19 provides an example of applying the bubble-correction methods ofthe present invention to provide accurate prediction of yeastconcentration.

FIG. 20 provides an example of applying the bubble-correction methods ofthe present invention to provide accurate prediction of e. coliconcentration.

FIG. 21 provides an example of a bubble calibration determined underconditions where variable concentrations of albumin are present inaddition to yeast biomass.

FIG. 23 provides an example embodiment of a schematic of the presentinvention providing the capability of monitoring the biomass in multiplevessels simultaneously.

FIG. 24 shows reflectance signal histograms for uncoated and titaniumnitride coated probes immersed in an aqueous medium containing yeastthat is being stirred and sparged with room air.

LIST OF COMPONENTS DEPICTED IN FIGS.

-   1—Light source-   2—Detector-   3—Mirror-   4—Beam splitter-   5—Optical fiber-   6—Sensor housing-   7—Sensor face-   8—Test tube-   9—Rear support-   10—Rail-   11—Spring-   12—Bottom aperture-   13—Sattering reference-   14—Support shaft-   15—Turning handle-   16—Base-   17—Strain relief-   18—optical fiber-   19—Multi-mode optical fiber

Detailed Description

The detailed description set forth below in connection with the appendeddrawings is intended merely as a description of the presently preferredembodiments of the invention, and is not intended to represent the onlyform in which the present invention may be constructed or utilized. Thedescription sets forth the functions and sequence of steps forconstruction and implementation of the invention in connection with theillustrated embodiments. It is to be understood, however, that the sameor equivalent functions and sequences may be accomplished by differentembodiments as suggested in the present description, and may besatisfactorily applied for the measurement of any material which mayexhibit similar behavior that are also intended to be encompassed withinthe spirit and scope of the invention.

The detailed description set forth herein will make reference to themeasurement of biomass in a liquid culture. The term “biomass” as usedin this patent application, refers to the concentration of biologicalmaterial, such as cells or microorganisms. What is meant here andelsewhere in the patent application by “concentration” is the number ofa type of particle, weight of a type of material, or volume of a type ofmaterial found in a given volume or weight of a medium.

Optical Density (OD) is defined as:

${OD} = {{- F}\; {\log_{10}\left( \frac{I}{I_{0}} \right)}}$

where light intensities are measured after transmission through a mediumcontaining suspended particulate matter (I) and through the same mediumin the absence of particulate matter (I₀). It is common in thebiofermentation field to measure and refer to biomass according to theoptical density measured at a particular wavelength, such as 600 nm(“OD600”), through a 1 cm path length cuvette, with a commercialspectrophotometer. When measuring OD using a spectrophotometer it isnecessary to dilute the sample to have an OD in a linear range ofresponse (commonly OD<1, but more ideally OD between 0.05 and 0.2) andthen scale the measured OD by the dilution factor, F. This type ofmeasurement is herein referred to as “Offline OD” measurement, todistinguish it from the real-time (“Online”) measurement allowed by themethods and devices for the present invention. For mono-disperse cells alinear relationship between biomass and OD generally holds.

The method(s) and instrument(s) of the present invention may also findapplication in liquid suspensions of solids other than biomass as wellas in solutions. For example, the particulate content in milk, the rateof polymerization in a chemical system or the turbidity of water may bemeasured by application of the method(s) and/or instrument(s) of thepresent invention. Similarly, the present invention may be utilized todetermine the amount of gas in a liquid phase, such as the concentrationof gas bubbles in a liquid medium. In addition, the attenuation ofradiation by absorption may be used to measure the concentration ofcomponents dissolved in solution, by application of the presentinvention.

The method and instruments of the present invention may also be useablein the gas phase. For example, in industrial plants using smokestacks,the amount or concentration of a specific component of the effluent gasmay be measured by application of the present invention. As anotherexample, the present invention may be used to measure the particulatecontent of a gas for the purpose of smoke or fire detection. As yetanother example, the present invention may be used to measure theconcentration of a particular component of a gas, such as theconcentration of carbon dioxide in a mixture of gases or the density offog or smoke in the flight path of an airplane.

In addition, the method(s) and/or instrument(s) of the present inventionmay be utilized to monitor materials in the solid state and to monitortransformation of materials between states. For example, the presentinvention may be used to monitor the conversion of a liquid to the solidstate, such as gel formation, or crystallization. Thus, although thehereinafter-set-forth descriptions often refer specifically to themeasurement of the biomass in a liquid culture, it will be appreciatedthat the method(s) and instrument(s) of the present invention are alsoapplicable in other liquids and in gas and solid media applications.

Description of Exemplary Methods

The present methods are generally directed to techniques of illuminatinga particulate suspension and determining the particulate concentrationin correlation to the amount of back-scattered light. Methods forreducing or avoiding the influence of potential interferents are alsoprovided. Such sources of interference may include objects submergedwithin the medium, such as other sensors, agitators, sparge tubes, andstirring baffles, as well as the wall of the container holding themedium. Another source of interference may be the presence of one ormore additional particle types, such as bubbles, or un-dissolved mediaconstituents.

In many embodiments, the methods comprise using an optical sensor todetermine the concentration of particles in a liquid medium held withina container. The medium is irradiated with a light source in the sensor.The light source has an emission wavelength that is absorbed by themedium, as characterized by a mean absorption path length. The sensoralso includes one or more detectors that detect source light scatteredby particles in the medium. In many embodiments, the spatial separationbetween the central optical axes of the light source and detector at theoptical interface with the medium is substantially less than the meanabsorption path length. At the same time, the distance between theoptical interface with the medium and the nearest potentiallyinterfering stationary object falling within the emission cone of thelight source, is substantially greater than the mean absorption pathlength. The nearest stationary object can be considered not to include acontainer wall on the container side through which the media is beingilluminated and/or detected. The detected light is then correlated tothe concentration of particles in the medium.

In embodiments where the medium is aqueous, absorption of the sourcelight by water may influence the mean absorption path length (1/μ_(a)).The mean absorption path length will in turn influence the maximumoptical penetration depth—the maximum depth into the medium that thesource may penetrate before being attenuated below the detection limit.In some embodiments the source wavelength consists of infrared lightthat is absorbed by the medium. An example of the influence of theinfrared light source wavelength on the optical penetration depth isprovided in Example 1 and depicted in FIG. 2. In this example, threenear infrared source wavelengths, having increasing water absorptionwere selected: 980 nm (S1,1/μ_(a)=21 mm), 1310 nm S2, 1/μ_(a)=6 mm), and1550 nm (S3, 1/μ_(a)=0.8 mm). Back-reflected light was collected at twodistances from the source (D1˜1 mm, D3˜7 mm) with the sensor immersed inbiomass at a range of concentrations (yeast: 0.1-100 g/L). As shown inFIG. 2, at the lowest biomass concentration, the optical penetrationdepth at 980 nm (S1), where water absorption is relatively weak, canextend to about 90 mm. At 1550 nm (S3), where the water absorption isrelatively strong, the penetration depth is limited to about 6 mm. At1310 nm (S2), where the water absorption is intermediate between that at980 and 1550 nm, the penetration depth is about 30 mm. Note that thepenetration depth is also influenced to some extent by thesource-detector separation, but in this example the choice of sourcewavelength has a stronger influence. The maximum optical penetrationdepth is also influenced by the particle concentration in the medium,with the penetration depth typically decreasing with increasing particleconcentration, such as shown in FIG. 2.

The maximum optical penetration can alternately be considered as theminimum allowable distance between the optical interface of the sensorwith the medium and any potentially interfering objects in the vesselcontaining the medium (which may include the vessel itself). In manyembodiments it will be beneficial to choose the source wavelength sothat the distance to potentially interfering objects is at least twicethat mean absorption path length. In other embodiments, higher multiplesof the mean absorption path length may be used to determine the minimuminterference distance. In Example 1, mean absorption path lengthmultiples of about 4, 5, and 7, respectively for source wavelengths of980, 1310, and 1550 nm, are appropriate for selecting the minimumdistance to potentially interfering objects in the medium, when it isdesired to determine particle concentration in the lowest concentrationrange. This path length multiplier can be decreased if the range ofbiomass concentrations to be measured is restricted to a higherconcentration range. In Example 1, if the biomass concentrationmeasurement range is restricted to above 1 g/L or above 10 g/L, anappropriate multiplier may be in the range of 3-5 or 1-3, respectively.

In some embodiments of the invention a collar is placed on an immersibleprobe to aid the user in achieving a pre-determined depth in the vesselduring assembly of the probe onto the vessel. By inserting the probe tothe depth defined by the collar, the user is ensured of immersing theprobe into the medium, but is prevented from accidentally placing theprobe in too close proximity to the bottom of the vessel. In manyembodiments the position of this collar is adjustable, according to thevessel size being used. The collar position is pre-set duringmanufacturer for use on a particular vessel type but may be adjusted bythe user for other vessel types. In some embodiments markings areprovided on the probe to indicate the collar position that is mostappropriate for particular vessels types or sizes.

The choice of the source-detector separation can have a strong influenceon the range of particle concentration that is measureable. Byconfiguring the sensor so that the separation between the light sourceand detector is small compared to the mean absorption path length in themedium, a wide range of particle concentrations may be measureable withthe same source-detector pair. Further, the linearity of the measuredreflectance signal may be increased by keeping the source-detectorseparation small. Measurements at two source wavelengths (1310 and 1550nm) and several source-detector separations (˜0.2, ˜1, and ˜3 mm) aredescribed in Example 2. As shown in FIG. 3, when the source-detectorseparation was chosen to be comparable to the mean absorption pathlength (S1-D3 and S2-D2), the range of sensitivity to changes in biomassconcentration was limited compared to those configurations where thesource-detector separation was selected to be substantially less thanthe mean absorption path length (e.g. S1-D1 or S2-D1). In manyembodiments, in order to achieve the widest range of biomasssensitivity, it will be beneficial to select the source-detectorseparation to be at least two times smaller than the mean absorptionpath length. In other embodiments even higher factors may be beneficial.In Example 2, the configurations showing the widest range of sensitivityto biomass changes, S1-D1 and S2-D1, had source-detector separationsthat were, respectively, 30 and 4 times smaller than the mean absorptionpath length.

In some embodiments, the method comprises determining the concentrationin a medium of a first particle type in the presence of a secondparticle type. The method includes the steps of passing lightoriginating from a first light source into the medium, and detecting afirst light signal originating from the first light source that wasreflected from within the medium. Next, a first statistical measure iscomputed that is related to the central value of the signal, wherein thestatistical measure has reduced dependence on outliers compared to themean of the signal. The first statistical measure is then used todetermine the concentration of the first particle type in the medium,with substantially reduced dependence of the concentration of the secondparticle type compared to that obtained by using the mean as thestatistical measure.

Examples of statistical measures with reduced dependence on outlierscompared to the mean include the trimmed mean, mode, and median. Thetrimmed mean is computed by sorting the data, discarding upper and lowerranges of the sorted data, and computing the mean of the remainingpoints. The trimming percentage may vary according to the application,but as an example, the upper and lower quartile of the data may bediscarded (“25% trimmed mean”) before computing the mean. The mode maybe computed as a single mode or as multiple modes. In such cases, themode value that is selected may depend on the application. In manyapplications the minimum mode value in a multi-mode distribution isselected. In many applications the number of bins in the histogram usedto compute the mode may also be variable. As an example, the number ofbins may be adjusted according different statistical estimates of thedistribution and central value of the data. As described in Example 4,one method for determining the number of bins is to divide the fullrange by the interquartile range with appropriate scaling andoffsetting. The interquartile range is the difference between the valuesof the data points at the 25^(th) and 75^(th) percentiles of a sorteddata set.

An example of the reduced dependence of biomass estimation on theinfluence of bubbles based on statistical measures other than the meanis provided in Example 4. The data depicted in FIGS. 5d and 5e werecollected over a wide range of yeast concentrations, agitation rates,and sparge (bubbling) rates. Each different biomass concentration isdepicted with a different symbol. Comparison of the mean values in FIG.5d with the median values in FIG. 5e , make it evident that the medianvalue at each biomass is less affected by changes in agitation andsparging, than is the mean value. This is particularly evident in thelow biomass range (<1 g/L). For example compare the range of mean valuesobserved at a biomass value of 0.2 g/L in FIG. 5d , to the range ofmedian values for the same biomass in FIG. 5e . When using the mean asthe central value measure, bubbling has the effect of smearing the data,so that 0.2 g/L cannot be distinguished from 0.1 or 0.4 g/L biomassvalues. In contrast, when using the median as the central value measure,the biomass points at 0.2 g/L are tightly clustered under all agitationand bubbling conditions, and are readily distinguished and separatedfrom those at 0.1 or 0.4 g/L.

In another exemplary embodiment, the method determines the concentrationin a medium of a first particle type in the presence of a secondparticle type. The method includes the steps of passing lightoriginating from a first light source into the medium, and detecting afirst light signal originating from the first light source that wasreflected from within the medium. Next, a first statistical measure iscomputed that is related to the central value of the signal, and asecond statistical measure is computed that is related to thedistribution of the signal. The first and second statistical measuresare then combined to determine the concentration of the first particletype in the medium, and is substantially independent of theconcentration of the second particle type.

Examples of statistical measures related to the central value of thesignal include the mean, trimmed mean, mode, and median. Examples ofstatistical measures related to the distribution of the signal includethe standard deviation, range, interquartile range, mean absolutedeviation, median absolute deviation, and mode absolute deviation.Examples of the reduced dependence of biomass estimation on theinfluence of bubbles based on combining statistical measures of thedistribution and central value are provided in Example 4. For example,FIG. 5e depicts the median absolute deviation plotted versus the mediansignal over a wide range of biomass concentrations, agitation rates, andsparging rates. The points at each biomass level are well clustered onthe plot, so that the biomass can be distinguished despite the influenceof bubbles. Measurement of biomass that is substantially independent ofbubble influence can therefore be accomplished by mapping of the biomassrange onto a plot of signal distribution and central value. In someembodiments this mapping consists of a series of lines used tocharacterize each biomass value. In other embodiments, one or moreanalytical functions are empirically derived that characterize thebiomass as a function of the central value and distribution. In one suchembodiment, two analytical functions are empirically derived, one thatcharacterizes the slope and another that characterizes the intercept ofthe lines through each biomass value.

In some embodiments, the mapping is used to generate an estimate of the“bubble-free” reflectance. One such embodiment is described in Example7. In this embodiment, the calibration consists of a series of lines inMAD-Median space. As shown in FIG. 15, the lines can be extrapolatedabove and/or below the calibration measurements, in order to accommodatemeasurements slightly beyond the bounds of the original calibration. Thecalibration is stored in instrument memory and is applied to newmeasurements by locating the nearest 2 calibration lines on the map tothe new measurement and interpolating between them to create a line onthe map that intersects with the new measurement. By tracing theinterpolated line down to it's minimum median value, the “bubble-freereflectance” is thereby determined. This minimum median reflectance canbe either the minimum of the extrapolated line, or the minimum of theun-extrapolated line. In a preferred embodiment, the minimum of theun-extrapolated line is used. By this method, the effect of bubbles onthe reflectance are largely removed, as shown in FIG. 16.

In other embodiments, such as described in Example 10 and depicted inFIG. 21, the bubble calibration map is more accurately represented by aseries of curves. In this case the bubble-free reflectance is determinedby tracing the curve down to the minimum median reflectance, inanalogous fashion to the procedure described for linear mapping. In someembodiments the curve is represented by a polynomial function. In yetfurther embodiments, the bubble calibration consists of a contour map inmedian-MAD space, and the bubble-free reflectance is determined byinterpolating between the contours nearest to the measured median andMAD values, and determining the minimum median reflectance of theresultant contour.

In some embodiments, the concentration in a medium of a first particletype is determined in the presence of a second particle type, with thedetection bandwidth and the measurement volume selected so that lightreflected from the medium allows signal fluctuations from the secondparticle type to be resolved, whereas the signal due to the firstparticle type is substantially constant at a given concentration. Byseparating the signal fluctuations from the constant signal, theconcentration of the first particle type is determined.

In some embodiments the medium is aqueous, and the first particle typeis biomass. The second particle type includes gas bubbles. In manyapplications the gas bubbles are room air, oxygen-enriched air, or acontrolled mixture of gases. The medium may be agitated at variablespeeds which may also have the result of introducing bubbles into themedium. The number and size of the bubbles suspended in the medium maybe affected by the rate of agitation as well as the concentration ofother particles suspended in the medium. In other embodiments the secondparticle type includes un-dissolved media constituents. Of particularinterest in the biofuels field, are bio-fermentation processes optimizedto break down cellulosic materials. The present invention providesmethods for distinguishing the live biomass that is breaking down thematerial from the material itself.

The effect of air bubbles on measurements of optical reflectance in aliquid biomass culture is described in Example 3. By selecting a closespacing between the source and detector and keeping the source anddetector apertures small (S1-D1 or S2-D1 in FIG. 1b ), the number of gasbubbles within the measurement volume is kept low enough so thatstatistical variation is readily observable. At the same time, thebiomass (yeast) particles used in this example were much smaller andmore numerous than the bubble particles, so that statistical variationdue to the biomass particles was low. The increased statisticalfluctuations due to the bubbles is demonstrated in FIGS. 4a and 4c ,which are measurements at the same biomass concentration (25 g/L yeast)with bubbling off and high, respectively. Power spectra of reflectanceamplitude vs frequency (FIGS. 6a-c ) show that in order to resolve thevariation due to bubbles moving in and out of the measurement volume, adetection of bandwidth of at least about 250 Hz or higher is necessary,in this example. For larger particles, such as media comprised ofcellulosic debris, the bandwidth necessary to resolve the particles maybe lower, such as 100 Hz. For very large particles, bandwidths as low as10 Hz may suffice.

The measurement volume necessary to resolve statistical fluctuations dueto the second particle type will also vary according to the particletype. Measurement volumes in the range of 1 μL to 1 L may allow bubblefluctuations to be observed. Table 0 summarizes the maximum measurementvolumes for the sensor depicted in FIG. 1a . The maximum measurementvolumes were estimated from the experimentally measured maximumpenetration depths (Example 1). As can be seen in Table 0, themeasurement volumes vary strongly according to the source wavelength andthe scattering properties of the medium, and more weakly according tothe source-detector spacing. The maximum measurement volumes in Table 0range between 4 μL and 200 mL. Bubble discrimination was found to bemore effective when employing source wavelengths at 1310 and 1550 nmthan at 980 nm. This is likely due to the smaller measurement volumeobserved when employing wavelengths that are more strongly absorbed bythe medium (e.g. water). When only the 1310 and 1550 nm sourcewavelengths are considered, the range of maximum measurement volumes isabout 4 μL to 20 mL.

TABLE 0 Estimated measurement volumes Source Max. Max. Max. Max. Wave-S-D Depth, Depth, Volume, Volume, length Spacing low scatter highscatter low scatter high scatter (nm) (mm) (mm) (mm) (mL) (μL) 980 1 754 200 30 980 7 110 6 700 100 1310 1 32 3 20 10 1310 7 35 5 20 70 1550 16 2 0.1 4

The measurement volume is most critical when the concentration of thefirst particle type is low. For example, when the first particle type isbiomass, and the biomass concentration is low, the measurement volumecan balloon to large volumes, unless a source wavelength is chosen thatis strongly absorbed by the medium. As shown in Table 0, with the sourcewavelength at 980 nm, where water absorbance is relatively weak, themaximum measurement volume is nearly 1 L when the biomass (i.e. scatter)is low. At wavelengths shorter than 980 nm, the measurement volume caneasily exceed 1 L. With such a large measurement volume the number ofbubbles within the volume can be large enough so that it is difficult toobserve fluctuations in the number of bubbles as a function of time. Incontrast, more effective bubble discrimination is observed at lowbiomass, when a strongly absorbing source wavelength is selected. Asshown in Table 0, at source wavelength of 1310 nm, the absorbance of thesource by water limits the maximum measurement volume to about 20 mL,even when the biomass (i.e. scattering) is low. Yet further restrictionin the measurement volume is achieved at 1550 nm, where the measurementvolume is limited to about 100 μL. Such a small measurement volume makesit easier to capture fluctuations as bubbles move in and out of theoptical field.

In typical embodiments, the light is infrared, the container is aculture flask, the media is aqueous, and the particles are cells. Thelight source and detector are typically arranged in a sensor housingacross a surface and aligned so the light is transmitted in a pathdescribing an acute angle from the direction of detector detection(e.g., optical axis of a conical detection zone for the detector).

In some embodiments the sensor is directly immersed in the medium, suchas described in Examples 1-5 and 8. In other embodiments the sensor ispositioned outside of the container (“non-invasive”), such as describedin Examples 6 and 7. In such embodiments, methods are provided tominimize or eliminate the detection of reflections from the surface ofthe container itself (“specular reflections”). Such methods includeincreasing the separation between the source and detector and limitingthe numerical and physical aperture of the source and detector. Asdescribed above, in many applications it is important to minimize themeasurement volume, in order to discriminate against a second particletype, and/or to maximize the range of sensitivity to changes inconcentration of a first particle type. In such applications, it isimportant to balance the need to avoid specular reflections withoutsacrificing sensor performance. This may be accomplished by configuringthe sensor geometry according to the properties of the container.Example 6 describes optimization of sensor geometry according to thethickness of the container wall. Referring to FIG. 8 and Table 6,source-detector overlap through a container wall of thickness, T, wasoptimized by varying the physical apertures (D₁ and D₂), numericalapertures (NA₁ and NA₂), and separation (Y) between the source anddetector. Example 7 describes experimental findings for one such sensor,designed for making non-invasive measurements through the 1 mm thickwall of a plastic vessel.

In other non-invasive embodiments, specular reflections are minimizedthrough the use of crossed polarizers. In one such embodiment, a firstlinear polarizer is positioned in front of the light source, and asecond linear polarizer is positioned in front of the detector. Thefirst and second linear polarizers are oriented so that theirpolarization axes are substantially perpendicular. In this manner, lightundergoing a single reflection (such as specular reflections) will beblocked by the second linear polarizer, whereas light that undergoesmultiple reflections and is thereby de-polarized (“diffusereflections”), will partially pass through the second linear polarizer.In other embodiments, a single polarizing beam splitter is used toaccomplish the polarization discrimination. FIG. 10c depicts one suchembodiment, in which the source light, 1, passes through the polarizingbeam splitter, 4, on its way to the sample. Light reflected back fromthe vessel wall that retains its original polarization state will passback through the polarizing beam splitter, 4, avoiding detection.However, light that is depolarized, such as by multiple reflections fromparticles suspended in the medium, will be partially-reflected by thepolarizing beam splitter, 4, allowing it to reach the detector, 2.

The container of media is typically a container designed to hold orstore media, but not typically a container normally employed to functionwith spectroscopic equipment in, e.g., a quantitative analysis. Thecontainers in the present methods are typically, e.g., a shaker flask, aT-flask, a centrifuge tube, a test tube, a roller bottle, a fermentor, abioreactor, a stir flask, a carboy, a bag, a media bottle, a multiwellplate, a petri dish, a syringe, a pipette and/or the like. In a typicalembodiment, the container is not a cuvette (e.g., from aspectrophotometer or fluorometer) and is not a spectroscopic flow cell(e.g., from an assay device).

Where the media is aqueous, the light wavelength is typically aninfrared (IR) wavelength. This is because IR wavelengths are wellscattered by particles typically of interest and such wavelengths aresubstantially absorbed by shallow depths of water, with the benefitsdescribed above. The light source wavelength in the methods can alsodepend to some extent on the optical qualities of the container wall ina particular instance. Other useful ranges of light source wavelengthsrange from less than 650 nm to more than 2000 nm, from about 700 nm toabout 1500 nm, from 800 nm to about 1300 nm. In certain embodiments thepreferred wavelength is about 1550 nm. In other applications the lightsources emits light between 1150 and 1350 nm, between 920 and 1150 nm,between 1350 and 1900 nm or about 1500 nm. In the context of othermedia, such as organic solvents, other optimum interrogation wavelengthsare available, e.g., wherein the particles of interest scatter the lightwell, and the solvent limits penetration of the light to desired shallowdepths.

In some of the methods, it is useful to have the light source anddetector on the same side of the container for the analysis. In thisway, the scattered light path is not dependent on the container toprovide consistent and adequate geometry. That is, a significant benefitof the present methods is that particle detection does not depend on theshape or size of the container holding the media. For example, as longas the media to be tested is in contact with the inside wall of thecontainer at a certain position, the methods allow the reading device torealize an appropriate interaction with the media. The media does nothave to be withdrawn or diluted. It matters little what is the shape orsize of the container or whether the container is full. In manyembodiments, the detector and light source are mounted in a devicesensor with their objective aperture in a common plane and directed insubstantially the same direction. In preferred embodiments, the lightsource and detector are arranged so that light emanating from the lightsource is scattered by particles in the media and returned to thedetector describing an angle of from less than 5 degrees to 45 degrees,from 10 degrees to 35, from 15 degrees to 30 degrees or about 25degrees.

The light source may radiate light in, e.g., a beam or cone, and thedetection zone visible to the detector can be described by, e.g., acylinder or cone. In preferred embodiments, the optical axis of thelight source and the optical axis of the detector detection zone aresubstantially parallel. In other embodiments the axes can converge ordiverge from each other at an angle ranging from about 1 degree to 45degrees, from 3 degrees to 30 degrees, from 5 degrees to 20 degrees orabout 10 degrees.

The particles detected in the methods can be any of interest, e.g., in areasonably uniform suspension. The particles of interest are typicallyfrom the fields of materials science or biological sciences. Typicalparticles of interest in the present methods include, e.g., bacteria,fungi, animal cells, plant cells, polymer particles, nanoparticles, solgels, proteins, viruses, and the like.

The methods include means of positioning and confirming the position ofa light-detector sensor system in relation to the container and media ofinterest. Uniform and optimal positioning of the sensor can be importantto the precision, accuracy, sensitivity and consistency of particleconcentration measurements in the methods. Position sensors andalignment guides of the inventive devices are discussed at length below.

The methods include provision of two or more detectors in functionalrelation to one or more light sources. In the methods, positioning ofthe sensor, uniformity of the media components, uniformity of the mediadepth and uniformity of the container wall can be suggested bycomparison of signals returned from two or more detectors at differentpositions. The detectors can be positioned symmetrically orasymmetrically relative to the light source. For example, twosymmetrically positioned detectors elements can be positioned to providea means of determining whether sufficient medium is present in order tomake an accurate measurement of particle concentration. Comparison oftwo symmetrically positioned detectors can also provide a means ofidentifying nearby interferences. For example, a bubble residing on thesensor may interfere with the optical measurement, but will typicallyinterfere to a different extent with two symmetrically placed detectors.By rejecting measurements for which the detector reflectances are not inagreement, such interference may be avoided. As another example, thereflectance from a nearby object in a bioreactor such as an impeller,sparge tube, baffle, or another sensor may cause interference with thesensor measurement. In some embodiments, disagreement between thereflectance measured by the two or more symmetrically placed detectorsleads to an error message being reported to the user that the sensor orprobe needs to be repositioned.

In the methods, two or more sources having different emissionwavelengths may be combined within the same sensor. Fiber opticembodiments of such sensors are depicted in FIGS. 1a and 1b . Free-space(i.e. non-fiber-optic) embodiments are depicted in FIGS. 10b and 10d .The combining of the multiple source wavelengths may provide the benefitof: (1) extending the measurement range of particle concentrations, (2)improving the discrimination against a second particle type, (3)providing a means of determining whether sufficient medium is present inorder to make accurate measurement of particle concentration, and/or (4)allowing particle concentration to be determined over a wider range ofmedium volumes.

In some embodiments, the two of more source wavelengths have differentmean absorption path lengths in the medium. In one such embodiment,first and second source wavelengths are provided at 1310 and 1550 nm,respectively, and the sensor is used to measure biomass, and bubbles mayalso be present as a potential interferent to the measurement. Due tothe longer mean absorption path length in water, the measurement volumeat 1310 nm will be greater than that at 1550 nm. As a result, themeasurement sensitivity at low biomass may be better at 1310 nm, whereasat 1550 nm the high range sensitivity linearity may be better (forexample, see FIG. 3). For the purpose of bubble discrimination, 1310 nmmay provide better data clustering in the low biomass range, whereas1550 nm may be better in the medium to high range (compare FIGS. 5e and5f ). Measurements made at 1310 nm will have a maximum penetration depthof about 30 mm, whereas at 1550 nm the depth will be limited to about 6mm (see FIG. 2). Disagreement of two separate biomass concentrationmeasurements based on the 2 source wavelengths, may therefore beindicative of insufficient fluid being present or the presence of aninterfering object in front of the sensor. In other applications, themeasurement-volume may be selected by the instrument operator, so thatthe appropriate source wavelength is used for the measurement.

Once scattered light is returned to the detector, the associated signalcan be correlated with other useful parameters such as optical density(OD) values or particle concentration. The methods can optionally beused to quantitate the amounts of the particles of interest present inthe sample. For example, in one class of embodiments, an intensity of asignal scattered back to the detector is measured and correlated (e.g.,through a standard formula determined through regression analysis) witha quantity of the corresponding particles of interest present. Thestandard formula can then be used to calculate an unknown amount ofparticles in a sample based on the output signal intensity for thatsample. Demonstrations of converting reflectance signal intensity intobiomass (dry cell weight) are provided in Examples 7-9.

To increase the precision and accuracy of measurements, the light sourcecan be monitored with a sensor that feeds back to the controller, whichinstructs compensating changes in radiant flux emitted by the lightsource, thus stabilizing the light irradiation. An example of laserfeedback control is provided in Example 7. Another way to enhance theaccuracy of particle concentrations in the methods is to take a blankreading of media without particles and subtracting the blank readingfrom a measurement of the media containing the particle.

In a particular embodiment, samples can be identified and trackedemploying components of the inventive device acting as a bar codescanner. For example, the light source can be a laser and lightreflected back to the detector during a sweeping motion across a barcode decal can be detected and interpreted by the processor to identifya sample.

Description of Device Embodiments

The devices of the invention are generally directed to one or more lightsources paired with one or more sensors arranged to interrogate a sampleof particles in a medium, with the sensor either immersed directly inthe medium, or positioned to measure through the walls from one side ofa container. For example, the devices for determining the concentrationof particles in a medium, can include a housing containing a sensor, alight source in the sensor, a detector in the sensor positioned todetect a signal of source light wavelengths scattered by particleswithin the medium, and a processor configured to correlate the detectedsignal to the concentration of particles. Typically the light wavelengthemitted by the source is selected to be absorbed without overlyextensive penetration into the medium. The device typically comprises acontroller configured to measure the detected signals and to control thelight sources.

It can be beneficial to make the light source distinctive overbackground light, and render it complimentary to certain detectorcircuitry. In a preferred embodiment, the light source of the device ismodulated in amplitude and/or frequency. In such a case, accuracy can beenhanced wherein the detector reading frequency is different from thelight interrogation frequency, e.g., thus avoiding problematic beatfrequencies, and other interference. In preferred embodiments thedetector signals measurement rate is at least 4-fold, 10-fold or100-fold, or more different (preferably greater), than the modulationrate of the light source. In certain embodiments, a quadrature slopecorrection may be employed as part of a detector demodulation algorithm.

In situations where multiple particulate concentration readings areexpected over some time period, it can be useful to affix thelight-detector sensor housing to the vessel, thereby providing thecapability of making multiple measurements without the need forreapplication of the sensor to the vessel. Typical vessels containingmedia and subject to particle concentration readings by the devices ofthe invention include, e.g., shaker flasks, a T-flasks, centrifugetubes, test tubes, roller bottles, fermentors, bioreactors, stir flasks,carboys, media bags, media bottles, multiwell plates, petri dishes,syringes, pipettes and the like.

The sensors of the devices can have complex arrangements of one or morelight sources paired with one or more detectors. For example, signalscan be combined from two or more paired source-detectors with twodifferent source-detector separations. The light source or detectors canbe fiber optical components, which are optically linked toelectro-optical components that are physically separated from thehousing, wherein the device further comprises one or more additionaldetectors in functional relation to the light source. The device caninclude a second light source with a light wavelength different from thelight wavelength of the first light source, e.g., to selectively detecta different particle type or compliment a different container or media.The light source wavelength can be selected according to the separationbetween the source and the detector, e.g., to tailor the light pathlength to the expected particle density or media absorbance.

In certain embodiments, the device is configured to provide particulardesired characteristics, For example, the device controller can beconfigured to collect detected signals at least every 0.10 seconds,thereby allowing measurement of variation in the amount of medium orparticles in front of the sensor as it varies over time. The processorcan be configured to distinguish signals depending on an amount ofmedium present at the container. The processor can be configured tocorrelate a ratio of signals collected in the presence of differentamounts of media sample to the particle concentration.

In preferred embodiments, the light source and detector are bothdirected in the same direction. In many embodiments, the source anddetector are aligned within 1 degree, 2 degrees, 5 degrees, 10 degrees20 degrees or 30 degrees of each other.

Sensor Optical Arrangement.

Embodiments of sensors of the present invention are illustrated in FIGS.1, 8, 10-12, 14, and 18. Referring to FIGS. 11a-d , a sensor housing, 6,holds one or more optical fibers, 5, and provides apertures into and outof the sensor. The numerical aperture of the optical fiber may beselected to limit the divergence of the light source and/or thecollection cone of the detectors. In some embodiments (e.g. FIG. 11a ),the same optical fiber, 5, is used both to deliver the source light andto collect the light for detection. In some such embodiments a beamsplitter may be used to combine and separate the source and detectionlight. In other such embodiments a fiber splitter (e.g. 2×1 branching)is used to combine and separate the source and detection light.

In other embodiments (e.g. FIGS. 11b-e ) separate optical fibers areused to conduct the illumination (source) and detected light. In thesimplest case (e.g. FIG. 11b ) one fiber is used to deliver the sourcelight, and a second fiber is used to collect the light for delivery tothe detector. In other embodiments, such as depicted in FIGS. 11cthrough 11e , one fiber is used to the deliver the light, and multiplefibers are used to collect the light for detection. In some suchembodiments the detection fibers are delivered to the same detector. Theadditional fibers used for light collection thereby increase the size ofthe detected signal, which may be important when the signal magnitude islow, such as at low biomass. In other embodiments, each fiber is coupledto a separate detector. The use of detection at multiple distances fromthe source fiber, may allow the range of biomass to be extended.Alternatively, the detector fibers may be symmetrically located aroundthe source fiber. By comparing the results from the multiple detectors,the presence of interfering objects, or the absence of sufficient fluidin front of the sensor may be determined. In this manner, the use ofmultiple symmetrically-located and separately-detected fibers may allowfor improved measurement accuracy and/or rejection of inaccurateresults.

The fiber optic embodiment depicted in FIG. 11e offers the benefit ofeasy and reproducible manufacture. A single hole may be made with adiameter just sufficient to accommodate the three fibers in aclose-packed triangular arrangement. During assembly the fibers areforced into an equilateral triangular arrangement by the constraintimposed by the hole diameter. The separation between the source anddetector fibers may be adjusted by selecting fibers having differentcladding diameters, or by leaving the fiber buffer layer in place, sothat it determines the close-packed diameter. In one particularembodiment, three fibers with 200/220/239 mm core/cladding/bufferdiameters (such as Polymicro part number FIP200220240) are used. Thefiber buffer is left in place during assembly, and the radius of thehole made to accommodate the three fibers has a radius of approximately520 μm.

In other sensor embodiments, provision of the light source and detectionare accomplished without the use of optical fibers. As with thefiber-optic based embodiments, such free-space embodiments may be usedeither non-invasively (i.e. measuring through the wall of the vessel),or invasively (i.e. immersed in the medium). Several free-spaceembodiments are depicted in FIGS. 10a-d . In FIG. 10a , the source anddetector are directed to and from the measurement medium through the useof mirrors, 3 a-c. The use of a two-sided right-angle mirror (e.g. 3 bin FIG. 10a ), allows the source and detector light to enter and theexit the medium as collinear beams with minimal separation. Theembodiment depicted in FIG. 10b , further includes a dichroic beamsplitter, 4, for combining the two light sources, 1 a and 1 b, into asingle beam, with high efficiency.

The free-space embodiment depicted in FIG. 10c employs a beam splitter,4, to combine the source and detection beams. The advantage of thisarrangement is that the source and detection beams can be overlapped tothe extent desired. Further, by using a polarizing beam splitter, thedetection of specular reflections from the vessel wall may be minimized,in applications where the sensor is used for non-invasive measurements.The embodiment depicted in FIG. 10d is an extension of that depicted inFIG. 10c , to include a second overlapped source wavelength.

In many embodiments of the invention, a laser monitoring detectorprovides a signal for measuring and/or controlling the laser radiantflux. The laser is directed into the medium either directly, or througha wall or aperture (“window”) of a vessel into a medium. The vesselwindow should be at least partially transparent to light at the laserwavelength. Suitable vessels include flasks, bottles, tubes, fermentors,and bioreactors with window material made from such opticallytransparent materials as plastic (e.g. polyethylene terephthalate (PET),polycarbonate (PC)), or glass. The preferred embodiment described hereis especially well-suited for thin-walled (<6 mm thick) vessels, such asis typically found in laboratory shake flasks, roller bottles, andtubes.

After the source light has penetrated the vessel window, cells ormicroorganisms within the medium scatter the light, some of which isreflected back towards the sensor. Reflected light is detected by atleast one reflectance detector. In some embodiments, the optical fibersare close-packed, so that the source-detector separation is determinedby the diameter of the cladding, or if the buffer is left intact, by thediameter of the buffer. In Example 8, described below, both the sourceand detection fiber have a core and cladding diameter of 0.2 and 0.22mm, respectively, and a numerical aperture of 0.22. The fibers areclose-packed, with the center-to-center distance between the laser andeach detector approximately 0.22 mm, as determined by the fiber diameter(with cladding).

In the embodiments depicted in FIGS. 11c to 11e , additional detectionfibers are provided. In one particular embodiment, the center-to-centerdistance between the source and each detector fiber is approximately0.22 mm and the numerical aperture of the fibers is 0.22. The numericalaperture of the fiber limits the full angle of the emission anddetection cones to about 25 degrees in air, or about 19 degrees inwater. Many other combinations of fiber diameters and numericalapertures could be used, depending on the desired measurement volume.For example, by selecting fiber with numerical aperture of 0.11, theangle of the detection and emission cones would be reduced to about 9degrees in water, thereby substantially reducing the measurement volume.In other embodiments the close-packing diameter is determined by thefiber buffer diameter. For example, in alternate version of theimmersible probe described in Example 8, the buffer is left in placeduring manufacture of the probe, thereby increasing the source-detectorseparation while still having the simplicity of a close-packedarrangement. For example, with a buffer diameter of 0.239 mm, the sourcedetector separation will be increased to 0.239 mm from 0.22 mm, bykeeping the buffer intact. One potential advantage of this increase insource-detector separation, is that sensitivity to dirt or fouling atthe sensor tip may be reduced.

In addition to being scattered by the contents of the medium, the laserwavelength is chosen so that it is partially absorbed by the mediumitself. Water, being the principle constituent of all cell media andrelatively invariant in concentration, is an ideal candidate forproviding this partial absorption of the light source. The absorption bythe medium needs to be low enough so that the light has a chance toscatter from cells in the medium and return to the detectors beforebeing absorbed. On the other hand, the absorption needs to be highenough so that light scattered from the cells and then reflected by thevessel wall, objects external to the vessel, or non-cellular objectswithin the vessel, has a low probability of returning to a locationwithin the vessel from which it can enter the detector apertures beforebeing absorbed by the medium.

The mean absorption path length of the medium at the source wavelengthwill determine the maximum distance between the source and reflectancedetectors at which the reflectance signal will be detectable. In mostapplications, the source-detector separation should be kept less than afactor of about ten times the mean absorption path length in order to bedetectable. In many applications, in order for the signal to beoptimally detected (e.g. with high signal to noise ratio), the factorshould be kept less than about 5, less than about 2, or in manyapplications less than about 1. For example, with a source wavelength of1310 nm, the mean absorption path length in water is about 6 mm. Atsource-detector separations of greater than about 60 mm, the signal willbe difficult to detect, and for optimal detection, the source-detectorseparation should be less than about 30 mm, less than about 12 mm, orfor many applications less than about 6 mm. As another example, at asource wavelength of 1550 nm, the mean absorption path length in wateris 0.8 mm. Signals at 1550 nm become difficult to detect atsource-detector separations greater than about 8 mm, and thesource-detector separation is optimally kept below 4 mm, about 1.6 mm,or in many applications, less than about 0.8 mm.

In addition to considerations of signal detection, the effect of thesource-detector separation on the range of sensitivity to particleconcentration changes should also be considered in many applications ofthe present invention. The selection of one or more source-detectorseparations optimized for measurement of particle concentrations inparticular concentration ranges has been described in prior art (e.g.U.S. Pat. No. 8,603,772). The unexpected finding of the presentinvention is that by selecting the source-detector separation to besubstantially less than the mean absorption path length in the medium,the range of sensitivity to particle concentration can be greatlyextended. In many applications requiring a wide range of sensitivity toparticle concentration, the source-detector separation should be keptless than the mean absorption path length, by a factor of about one half(½), a factor of about one fourth (¼), or a factor of about one tenth (1/10) times. For example, referring to FIG. 3 and Example 2, at a sourcewavelength of 1550 nm, with a source-detector separation that isapproximately equal to the mean absorption path length (0.8 mm), theexperimentally observed (red squares in FIG. 3) range of sensitivity tochanges in biomass is about two orders of magnitude (from about 1 to 100g/L yeast concentration). But by decreasing the source-detectorseparation to about 0.2 mm, or about a factor of one fourth (¼) timesthe mean absorbance path length, the range of sensitivity to changes inbiomass is extended to about four orders of magnitude (from about 0.02to 200 g/L yeast concentration). A similar pattern is observed with thesource wavelength set to 1310 nm. Again referring to Example 2 and FIG.3, with the source-detector separation chosen to approximately match themean absorption path length (about 6 mm), the range of sensitivity tobiomass change is limited to about two orders of magnitude (from about0.1 to 10 g/L yeast dry cell weight, data not shown). As thesource-detector separation is decreased to about a factor of ½ (3.2 mm),about a factor of ⅕ (1.2 mm), and about a factor of about 1/30 (0.2 mm)times the mean absorption path length, the range of sensitivity tobiomass change increases to about 3 (0.05 to 50 g/L), about 4 (0.01 to100 g/L), and more than 6 (about 0.0001 to 200 g/L) orders of magnitude,respectively.

Optimizing Sensor Geometry According to Vessel Wall (Window) Thickness

For applications in which the source and detected light must travelthrough a window on the way to and from the medium (e.g. non-invasivemeasurements through a vessel wall), the impact of the sensor geometryon sensitivity to window back-reflections may also need to beconsidered. If the source emission cone overlaps with the detection conewithin the window, there is a potential for interference of lightreflected from the inner wall of the window (“specular reflection”),with the desired light emanating from reflections off of particleswithin the medium (“diffuse reflection”). In general, increasing thesource-detector separation and decreasing the emission and detectioncone angles will lead to decreased interference due to specularreflections. However, since the range of sensitivity to changes inparticle concentration generally decreases with increasingsource-detector separation (as described above), the selection of theoptimal source-detector geometry may require a careful balance. In manyembodiments of the invention, the minimum source-detector separationthat still avoids significant interference from specular reflection isselected. Besides the source-detector separation, other factors that maybe varied to minimize the effect of specular reflections include: (1)the source and detection diameters, (2) the source and detectionnumerical apertures, (3) the central angle of incidence of the sourceand detector beams relative to the window surface, and (4) the relativepolarization of the source and detected beams.

Example 6 (below) describes the optimization of the sensor geometrybased on 2 of these factors, fiber diameter and numerical aperture, in asensor consisting of a single source and detection fiber, such asdepicted in FIG. 8, that is interfaced to a vessel having wallthickness, T. Configuration 1 in Table 6 corresponds to the case wherethe window thickness, T, is zero: the sensor is directly immersed in themedium. This configuration was experimentally tested, as described inExamples 1-5. In order to predict how these results could be extended tonon-invasive measurements, where the wall thickness (T) is greater thanzero, a simple geometrical model was developed which predicts the effectof source and detector diameter (D₁ and D₂), numerical aperture (NA₁ andNA₂), and edge-to-edge separation (Y), on the depth of optical overlapwithin the medium (X), and the closest point of intersection (Z) withinthe container wall. In configurations 2-16, several different wallthickness are considered. The same optical overlap depth as in case 1,is maintained by varying the source and detector diameters, numericalapertures, and edge-to-edge separations. By also maintaining Z as apositive number, sensitivity to specular reflections may be minimized.

In configuration 3 (Table 6), having window thickness 2.1 mm, the sameoptical fiber diameters and numerical apertures are used as inconfiguration 1, but the edge-to-edge separation between the fibers isincreased from 0.04 to 0.66 mm, in order to maintain the same opticaloverlap depth (X=0.71 mm). The model predicts that this can be donewhile still substantially avoiding specular reflections (Z>0). Sincethis optical model neglects the effects of scattering and multiplereflections within the window, in some applications it may provebeneficial to further separate the overlap of the source and detectorbeams within the window. This could be accomplished by decreasing thediameter and/or numerical aperture of the source and/or detector fibers.Configuration 2 describes the case where the diameter of both the sourceand detector fibers is decreased from 0.2 to 0.1 mm. By slightlyincreasing the edge-to-edge separation between the fibers (Y=0.76 mm)compared to configuration 2, the same optical overlap depth ismaintained (X=0.71 mm), but the separation between the source anddetector beams within the window is increased (from 0.04 to 0.14 mm).The effect of increasing the diameter of the source and detector fibersis shown in configurations 4 and 5 in Table 6. By doubling(configuration 4) or tripling (configuration 5) the fiber diameters,while still maintaining the same optical overlap depth (X=0.71 mm), thesource and detector beams will overlap within the container wall (Z <0),so that specular reflections may interfere with the desired measurementof diffuse reflections emanating from the medium.

In many applications it may be beneficial to use different diametersand/or numerical apertures for the source and detector. For example,when the source is a laser which can be coupled with high efficiencyinto a small diameter fiber with low numerical aperture, using a largerdiameter and/or numerical aperture for the detection fiber may providethe benefit of increased light collection, while still minimizingsensitivity to specular reflections. Examples are provided byconfigurations 6-10 in Table 6, where the diameter of the source fiber(0.1 mm) is chosen to be half that of the detection fiber (0.2 mm), andseveral different numerical apertures (0.22-0.48) are compared. Comparedto configurations 2 and 3, where the same source and detector diametersand numerical apertures are employed, configurations 7-10 are predictedto offer the benefit of reduced sensitivity to specular reflections(higher values of Z). Configurations 11-16 provide examples ofoptimizing the sensor geometry for several other window thicknesses.

Experimental results for a specific sensor embodiment designed formaking non-invasive biomass measurements through a 1 mm thick plasticvessel wall are described in Example 7. This sensor employs asingle-mode (approximately 0.01 mm core) fiber for the source light at1330 nm and a 0.2 mm core multi-mode fiber for detection. Thesource-detector separation of 0.7 mm (center to center) providessufficient discrimination against specular reflection from the vesselwall while still enabling linear biomass detection over more than 3orders of magnitude (see FIG. 17). Use of a single mode fiber for thesource provides high transmission efficiency of the 1330 nm laser diode,while allowing a low-cost fiber splitter to be used to deliver the lightto multiple sensors simultaneously.

In addition to source and detector diameter, numerical aperture, andseparation, the angle of the central optical axes of the source anddetection beams relative to the vessel window may also be adjusted tohelp reduce the influence of specular reflections on the measuredsignal. In some embodiments of the invention, the face of the sensor(e.g. component 7 in FIG. 11a ) is positioned so that it is not parallelto the surface of the window through which the measurement is made. Theextent of tilt may be selected according to the numerical apertureand/or separation of the source and/or detectors, and/or the windowthickness. A tilt angle of at least the half-angle determined by thenumerical aperture, may substantially reduce the effect of specularreflections. In other applications with greater sensitivity to specularreflections, tilt angles of at least twice, at least 3 times, or leastfive times the half-angle of the numerical aperture are selected. Forthe embodiment depicted in FIG. 11a , if an optical fiber having anumerical aperture 0.22 is selected, and the window material has anindex of refraction of 1.5, the half-angle of the numerical aperturewill be about 8 degrees. In this case, selecting a tilt angle of thesensor face, 7, relative to the surface of the window of at least 8degrees will substantially reduce the influence of specular reflectionson the measurement. Further increases in the tilt angle to at least 16degrees, at least 24 degrees, or at least 40 degrees, may further enablethe rejection of specular reflections in many applications.

In other embodiments of the invention, tilting of the optical axes ofthe source and detector are achieved by tilting the componentsthemselves, or by tilting the optical fiber through which the light istransmitted, rather than simply tilting the surface of the sensorrelative to the window surface. In such embodiments, the source anddetector optical axes may be tilted independently, to further optimizethe rejection of specular reflections in favor of diffuse reflectionsemanating from the medium.

Sensor Housing

For applications in which the sensor is directly immersed in the mediumto be measured, the composition of the sensor housing may be chosen soas to minimize interaction with the medium or the particles suspended inthe medium. In some embodiments, the sensor housing may be constructedfrom stainless steel, for example stainless steel type 316 L may besuitable for applications. The surface of the housing may also bepolished in order to minimize the potential for interactions with themedium. For example, a surface polish specification of N5 (<0.4 μm), maybe suitable when the medium contains biomass. In other applications, thesensor housing may be constructed from glass.

The adhesion of materials on or near the optical face of the sensor isminimized or eliminated in some embodiments of the present inventionthrough the selection of particular sensor face materials, the surfacepolish and coating of the materials, and/or the shape of the materials.In one embodiment, the tip of the sensor is coated with titanium orzirconium nitride. The effect of such a coating may be to reduce orprevent adhesion of gas bubbles and/or biological material such as cellsor protein, to the surface of the sensor. Reduced bubble adhesion totitanium nitride coated stainless steel compared to uncoated stainlesssteel is demonstrated below in Example 12. Zirconium nitride is known tohave similar properties to titanium nitride but has the benefit that itcan be applied (e.g. by vapor deposition) at lower temperatures (e.g. at200 C instead of 300 C) compared to titanium nitride. In otherembodiments the tip of the sensor is constructed from glass. In yetother embodiments, such as depicted in FIGS. 12b, 12c , and 18, thesurface of the sensor is rounded. The rounding of the surface, mayfurther aid in the reduction of adherent particles.

The front face of the sensor may be covered with a face plate thatprotects the sensor from the environment in which it is operated. Theface plate should be constructed from a material that is transmissive tolight emitted by the laser. In addition, the face plate may beabsorptive to light at wavelengths other than the laser emissionwavelength. An example of a suitable material for the face plate isEdmund Optic part number NT 43-954, which is transmissive to nearinfrared light but absorptive to visible light. In some embodiments agasket is used to prevent the sensor from sliding easily against thevessel surface. When pressed against a vessel wall, the gasket may alsobe used to create a seal that prevents materials from occluding oraffecting the optical measurement. In some embodiments, surrounding theface plate is a gasket groove used to contain the gasket. The gasketgroove helps to prevent the gasket from moving and possibly obstructingthe face plate.

Verification, Correction, and Calibration of Sensor Performance

In some embodiments of the invention, a material with a stablereflectance is provided for the purpose of checking, and if necessarycorrecting, the sensor performance. Examples of suitable materials forthis purpose are Kynar and Teflon. The expected reading may be stored ininstrument memory or written on the standard itself. If the actualsensor reading does not agree with the expected reading, in oneembodiment correction factors are applied to the reflectance measurementto bring it back into agreement. These correction factors may then bestored and applied to subsequent readings.

For sensor embodiments designed to be immersed in a medium, it may bedesirable, in some applications, to be able to perform the sensorverification and/or calibration step while the sensor is immersed in themedium. For example, when the sensor is immersed in a medium for growthof biomass that has been sterilized to prevent growth of undesiredorganisms, the sensor may need to be sterilized along with the vessel,precluding its subsequent removal, without jeopardizing the culture tocontamination. Sensor embodiments designed to allow thecalibration/verification step to occur without the need to remove thesensor from the medium, are depicted in FIGS. 12a through 12c. Thescattering reference material, 13, is attached to the sensor using asupport shaft, 14, that traverses the interior of the sensor housing, 6.A turning handle, 15, attached to the support shaft allows the user torotate the reference material into and out of the optical path of thesensor. FIGS. 12a and 12b depict embodiments with flat and roundedsensor faces, 7, with the scattering reference material positioned for averification/calibration measurement. FIG. 12c depicts the sameembodiment as 12 b, but with the scattering reference material rotatedout of the optical path, so that scattering can be measured fromparticles in the medium in which the sensor is immersed.

The sensor embodiments depicted in FIGS. 12a-c may have the addedbenefit of providing a mechanism by which interfering material may becleared from the front of the sensor face. For example, the accumulationof cells, cell debris, or proteins on the sensor window may interferewith the measurement of the particles suspended in the medium. In suchcases, the scattering reference material may function as a wiper, sothat one or more passes of the scattering material across the face ofthe sensor, may serve to clean it. As another example, in cases wherebubbles have a tendency to adhere to the face of the sensor, with thepotential to interfere with the diffuse scattering measurement, thescattering reference may be used to remove the bubbles from the sensorface.

Storage of Sensor Calibration Coefficients and Sensor Connector

In many embodiments it will be convenient to store coefficient valuesrelated to the sensor calibration or settings in the sensor itself. Inone embodiment, the sensor is an immersible sensor employing fiberoptics to transmit light between a base unit which contains theelectro-optical components (e.g. light sources and detectors) and thepoint of measurement in a medium. In order to allow the sensor to besterilized within the measurement vessel it will convenient in manycases to be able to disconnect the sensor from the base unit. A memorydevice, such as a 1-wire EEPROM, provides a convenient means for storingsensor calibration coefficients and settings. The memory device may belocated within the sensor housing, or within the connector on thesensor. In one embodiment the connector is a hybrid electro-opticalconnector that provides a mechanism for simultaneously connecting thefiber optic and electrical (e.g. memory device) components, within asingle connector. Such connectors are commercially available (e.g. F7connectors made by Lemo.

Data Acquisition Timing

A timing scheme for driving 2 lasers and reading one or more detectorsin a sensor of the present invention, is depicted in FIG. 9. The 2 lasersources are each driven at 1 KHz with a square wave pulse having 25%duty cycle. The relative phase of the lasers is fixed so that the “on”period of the 2 laser pulses are separated by about ⅙^(th) of amillisecond, to avoid any potential interference. The one or moredetectors are each read continuously at a rate of about 60 kHz, that issynchronized with the laser drive pulses. Dark periods (where bothlasers are turned off) are used to measure contaminating light levels(e.g. ambient light), which can then be subtracted from the signalsmeasured while the lasers are on, if necessary.

Test Tube Reader

An embodiment of the invention, designed for measuring particleconcentration in test tubes or other small vessels is depicted in FIGS.13a through 13c . The test tube, 8, is held in place on one side by arear support, 9, that has a V or U-shape in one dimension and issubstantially flat in a second dimension, so that it can stablyaccommodate test tubes having a range of diameters. The rear support isheld on a sliding rail, 10, that travels in a direction perpendicular tothe tangent of the test tube surface that is closest to the center ofthe V or U shape in the rear support. The rear support is urged againstthe wall of the test tube by a spring, 11, that links the rear supportand the base, 16, of the test tube reader. The side of the test tubeopposite the rear support interfaces with the sensor face, 7, where theoptical measurement is performed. In a preferred embodiment of theinvention, the sensor face includes a light source emitting atapproximately 1550 nm, and two symmetrically-placed detectors. The laserand detectors are positioned along a line that is parallel with the flatsurface of the test tube. The detectors are equally spaced from thelight source, with the center-to-center source-detector spacing betweenabout 0.1 and 3 mm, for example about 0.8 mm. In many applications itwill be convenient to use fiber optics to convey the light between theoptical elements (e.g. laser and detectors) and the sensor face. In manyembodiments, an aperture, 12, is provided in which the end of the testtube may be placed.

The choice of source wavelength will in part determine the minimum tubediameter on which accurate measurements of particle concentration may bemade. In the embodiment described above using a 1550 nm source, theminimum tube diameter will be about 6 mm, for measurements at lowparticle concentration (smaller tubes could be measured at higherconcentration). Selection of a source wavelength having a higherabsorbance than 1550 nm in the medium will allow accurate measurement intube diameters smaller than 6 mm. In some embodiments a stop is providedon the rail, which limits the motion of the rear support so that testtubes having a diameter smaller than a chosen value will not beaccommodated by the holder. For example, if the source wavelength is1550 nm, the stop may be positioned so that tubes having a diametersmaller than about 6 mm will not be held securely between the sensorface and the rear support. In yet further embodiments, at least onecontact sensor is provided, either on the sensor face or on the rearsupport, or both, that detects whether the test tube is being heldfirmly between the sensor face and rear support. Measurement isprevented unless the position sensor(s) are engaged. In this manner,measurement on tubes below the minimum diameter is prevented. Thecontact sensor also provides a mechanism for automating the measurementprocess: engagement of the contact sensor may provide a trigger signalto commence measurement.

In some embodiments, the contact sensor comprises at least onemechanical switch, which may be activated by contact pressure. In suchembodiments the position switches may be located on a printed circuitboard within the body of the sensor. Many suitable position switches areavailable, but one example is manufactured by Omron ElectronicComponents: part number B3U-1000P-B. This switch requires only 0.15 mmof travel with 150 grams of force in order to be activated, and has aconveniently small footprint (2.5×3.0×1.6 mm). A narrow rod may be usedto span the distance between the position switch and front of thesensor. One or more o-rings on the rod may be used to seal the cavityagainst moisture. This arrangement allows an inexpensive non-waterproofposition switch to be used, while still providing protection of theinstrument in wet environments.

In an alternate embodiment of the sensor, the position sensing isprovided by one or more capacitance sensors. The capacitance sensors mayfurther be selected and positioned so that they are only activated whensufficient medium is present beneath the surface of the window beingmeasured. In yet another embodiment of the sensor, the position sensingis provided by one or more pairs of light sources and detectors. Thesesource-detector pairs may be angled towards each other to detect surface(specular) reflections. The source divergence and detector acceptanceangles are restricted so that only when the vessel surface 103 is in thedesired orientation relative to the sensor, will a threshold level ofsurface reflection be detected.

In many embodiments, the apparatus is designed so that sensor face, 7,interfaces near the bottom of the test tube, so that tubes containingonly a small amount of medium may still be accurately measured. The useof 2 symmetrically placed detectors around the source provides a methodfor determining whether sufficient medium is in front of the sensor facefor accurate measurement, as further described in U.S. Pat. No.8,405,033. In an alternative embodiment, only a single detector isemployed.

In some embodiments a mechanism is provided for mixing the tubecontents, either prior to and/or during the optical measurement. In apreferred embodiment, the mixing mechanism is a shaker, such as a vortexmixer, that interfaces with the bottom of the tube. The mixing isautomatically initiated prior to the optical measurement to make surethat all particles are suspended and well-mixed, the mixer is turnedoff, and then the optical measurement commences. In addition to makingsure that the tube contents are well-mixed, the shaker may provide thebenefit of dislodging bubbles from the sides of the tube, that maypotentially interfere with the optical measurement. In some embodimentsa delay time is provided between the mixing period and the opticalmeasurement, in order to allow bubbles to escape from the fluid. In analternative embodiment, a magnetic stir bar is placed inside the tube,and a magnetically-coupled stirrer is provided near the bottom of thetube.

Alternative Embodiments

In some embodiments, the position of the laser(s) and detector(s) may beinterchanged with each other while still maintaining the essentialsensor functionality. For example, in the embodiments depicted in FIGS.11c-d , the single source fiber, 5 b, could be replaced with a detector,and the detection fibers replaced by two (FIG. 11c ) or more (FIG. 11d )source fibers. In order to separately measure the reflectance thatoriginated with each laser, the lasers may be turned on at differenttimes, or be modulated at different frequencies. When determiningmeasurement validity, the detected signals due to the two differentsources are compared, in an equivalent fashion to the manner in whichthe two detected signals emanating from the single source are comparedwhen employed by the sensors depicted in FIGS. 1a-b, 11c-e , and 18.

In other embodiments, additional lasers and/or detectors are added sothat there are source-detector pairs at multiple separation distances.One such embodiment is depicted in FIG. 11c , where optical fiber, 5 a,is coupled to a light source, and the 2 remaining fibers, 5 b and 5 c,are coupled to detectors. Detection fibers 5 b and 5 c may be separatelydetected and combined to extend the linear range response to particleconcentration. Optimal combinations of source-detector distances forachieving wide linear response to particle concentration, and algorithmsfor combining the signals from the different source-detector pairs aredescribed in Patent Application US 20090075248, which is included hereby reference.

In some embodiments the laser modulation is varied according to the sizeof the measured reflectance signal detected from the sample. In one suchembodiment, the amplitude of the laser modulation (“modulation depth”)is increased until a desired reflectance value has been obtained, or amaximum modulation depth has been reached. In this manner the dynamicrange of the measurement may be extended, for example allowing a widerrange of particle concentrations to be accurately determined.

In some embodiments an analog band-pass filter is added to the detectoramplification electronics, that selectively amplifies signals at thefrequency of the laser modulation, while attenuating signals outside ofthe band-pass range.

In some embodiments the reflectance signal is log-transformed twice andrelated to a singly log-transformed quantity related to particleconcentration, such as optical density. This relation may be a lineartransformation, higher order polynomial transformation, or othertransformation, such as described above. One advantage of some suchembodiments is that the relationship between the twice log-transformedreflectance signal to the singly log-transformed particle concentrationis linear over a wide range of particle concentrations. After the lineartransformation, the signal is inverse log transformed, in order to berepresentative of particle concentration.

In many of the embodiments described above, a light sources emitting inthe vicinity of 1310, 1330, or 1550 nm are described. However, lightsources emitting in many other spectral regions will also be suitable inthe present invention. The absorbance spectrum of water containsmultiple bands in the near infrared spectral region, with generallyincreasing absorbance peaks with increasing wavelength. The spectralregion may be selected according to the desired optical depth ofpenetration, with suitable spectral regions generally increasing inwavelength as the optical depth of penetration is decreased. Forexample, in applications requiring an optical depth of penetration inthe range of 1-10 cm, the 900-1150 nm spectral region is well suited.Water absorbance in this region ranges between about 0.06 and 1.0 cm⁻¹,corresponding to a range of mean absorbance path lengths from 1 to 16cm. One advantage of working in this spectral region is that numerouscommercial sources are available, and inexpensive low-noise detectorswith Silicon active areas may be employed. In another alternativeembodiment, a source in the vicinity of 1550 nm is employed. Suchsources have been extensively developed within the telecommunicationsindustry. The strong water absorbance in this spectral region (12 cm⁻¹),makes it well suited for short optical penetration depths, such as inthe mm range, particularly about 6 mm or less. In a further embodimentof the invention, multiple source wavelengths are employed in the samesensor, with different source wavelengths providing different opticalpenetration depths, so that the sensor can automatically adapt accordingto the available fluid depth in front of the sensor.

In many invention embodiments described herein, the physical separationand orientation between sources and detectors are used to diminish theinfluence of surface (specular) reflections on the measurement in favorof diffuse reflections from particles within a medium. In otherembodiments of the invention, additional or alternative methods are usedfor reducing the influence of specular reflections. Examples of suchmethods include: (1) the use of crossed-polarizers, (2) Angling of thesource and/or detectors, (3) high frequency (e.g. GHz) modulation anddetection, (4) the use of short light pulses and high speed detection,and (5) photo-acoustic measurement, where an acoustic detector is usedto detect and depth-resolve a light pulse.

In the description of some embodiments, a laser diode light source isdescribed. However, many other light sources could be substituted forthe laser diode without substantially modifying the essential featuresof the invention including: vertical cavity surface emitting lasers(VCSELs), light emitting diodes (LEDs), resonant cavity light emittingdiodes, solid state lasers (e.g. Nd-YAG), and gas lasers (e.g. HeNe).

EXAMPLE 1 Optical Penetration Depth as a Function of Source Wavelengthand Source-Detector Separation

An immersible fiber optic sensor was constructed and used to investigatethe maximum optical depth of penetration of light into an aqueousscattering medium. The body of the sensor was constructed from a hollowaluminum tube, 25 cm long, with 18 mm outer diameter. The end face ofthe sensor was constructed from black ABS, with holes drilled to guideplacement of optical fibers. Optical fibers were positioned flush withthe outer sensor face, glued into the place, and optically polished,resulting in a smooth, continuous external surface. The arrangement ofthe optical fiber on the end face of the sensor is depicted in FIG. 1a .S1-S3, and D1 are 200/220 μm diameter core/cladding fibers with 0.22 NA.D2 and D3 consist of 550/600 μm diameter core fibers with 0.22 NA. S1-S3are positioned so that they are close-packed along a line. The 7 fiberscomprising D3 are 6-around-1 close-packed. Laser diode light sourceswith three different wavelengths were coupled to three of the fibers:980 nm (S1), 1310 nm (S2), and 1550 nm (S3). All sources were laserdiodes operating at room temperature, modulated at 1 kHz with 25% dutycycle and peak power of 1-10 mW. The detector fibers (D1-D3) were eachcoupled into separate 1 mm InGaAs detectors. The detector amplifierspassed frequencies from DC to 100 kHz, and the gains were set between1.6×10³ and 1.6×10⁴ V/mW (the lower gain setting was necessary to avoidsignal saturation at biomass higher than about 50 g/L). The detectorsignals were digitized with 16 bit resolution at a sampling rate of 100kHz using a commericial multi-function data acquisition board (NationalInstrument USB-6341).

The sensor was suspended in a 1 L glass beaker, filled with Baker'syeast (Red Star Active Dry Yeast) suspended into 0.9% aqueous NaCl, atseven logarithmically spaced concentrations between 0.1 and 100 g/L. Amirror was placed at the bottom of the beaker, parallel to and directlybelow the optical face of the sensor. The distance between the frontface of the sensor and the front face of the mirror was measured. Thisdistance was increased until the signal dropped below the noise floor,and was recorded as the optical penetration depth.

FIG. 2 summarizes the measurements of optical penetration depth as afunction of yeast concentration for several different source-detectorpairs. The penetration depth depends strongly on the source wavelength,and more weakly on the distance separating the source and detectorfibers. The penetration depth determines the minimum fluid depthrequired for an accurate back-scattering measurement. Water absorbanceis weakest at the 980 nm (S1) source wavelength, and the opticalpenetration at low biomass is in the range of 65 to 90 mm. At 1310 nm(S2), where the water absorbance is between that at 980 and 1550 nm, thepenetration depth at low biomass is about 30 mm. With a 1550 nm source(S3) the penetration depth is limited to about 6 mm. As the biomassincreases the penetration depth decreases for all source wavelengths, sothat by 100 g/L the penetration depth is 10 mm or less in all cases.

EXAMPLE 2 Biomass Sensitivity Range without Bubbling

The same apparatus as in Example 1 was used to measure theback-reflectance signal as a function of biomass, but with the fiberoptic connections re-configured as shown in FIG. 1b . Only the 1310 and1550 nm sources were used, so that the third linearly close-packed fibercould be connected to a detector. Note that the three other detectionfibers/fiber bundles have been re-numbered accordingly. The sensor wasimmersed in a 12 L Magnferm (NBS) fermentor containing about 10 L ofmedia. The media consisted of 0.9% saline with dry active yeast (RedStar) suspended at 23 different concentrations ranging from 0.1 mg/L to200 g/L, in a geometric series, with series factor 2. The fermentor wasagitated at 400 RPM, and was not bubbled.

Each measurement consisted of 10 seconds of data acquisition (1×10⁶total data points were collected on each data channel). Data processingconsisted of: (1) demodulation of the detected signals according to thedifferent laser sources, (2) subtraction of the “laser off' signal fromeach of the “laser on” signals, and (3) normalization of each signal bythe source laser power and the detector gain. The first few points atthe start and end of each square wave laser pulse were removed, reducingthe number of measurement points within each laser pulse from 25 to 20.The result was (100 kHz)×(10 sec)×(25% duty cycle)×(20/25)=2×10⁵ datapoints for each source-detector pair. The central value of each data setwas estimated by computing the median.

FIG. 3 shows that in the absence of bubbling, by minimizing theseparation between the source and detector (S1-D1 and S3-D1), it ispossible to measure a monotonic change in back-scattering amplitude overan extremely wide range of biomass: 0.1 mg/L to 200 g/L, or more than 6orders of magnitude of biomass. This unexpected result demonstrates thatit is not necessary to use multiple source-detector separations toachieve a wide range of biomass sensitivity. Furthermore, due to thesmall size of the fibers (200 μm core) and their minimal separation(close-packed), the sensor size can be greatly reduced, compared toexisting commercially available immersible biomass sensors. With the D1detection fiber, both the 1310 and 1550 nm sources are capable ofmeasuring biomass over a very wide range, although the 1310 nm sourceproduced a more linear response with biomass. By including both sourcesin a sensor the biomass measured at 2 different depths could becompared, providing a means of rejecting measurements contaminated byreflections from non-biological objects or for which insufficient fluidis present in front of the sensor.

EXAMPLE 3 Effect of Bubbling on Reflectance Signals

The same apparatus and methods as used in Example 2 were used to collectmeasurement over a wide range of different agitation rates (0, 200, 400,600, 800, and 900 RPM) and air bubbling (0, 4, 8, 12, and 16 LPM). Dueto the high viscosity of the medium at very high biomass, bubbles takelonger to escape from the medium than at lower biomass, so that bubblesconsitute a higher fraction of the medium with increasing biomass. Forexample, with the agitation and sparge set to their maximum values, at abiomass of 100 g/L, bubbles consituted 54% of the total volume, whereasat 10 g/L biomass, bubbles only made up 7% of the total volume.

The S1-D1 (1310 nm laser, close-packed next to detector) signal in 25g/L yeast with sparging turned off, is shown in FIG. 4a . A histogram ofthe data (FIG. 4b ) shows that the distribution is reasonablysymmetrical, so that the mean (0.455), median (0.453), and mode (0.451)are all in close agreement. At the same yeast concentration, but withvigorous bubbling (see FIGS. 4c and 4d ), the distribution becomeshighly assymetrical, so that the mean (0.483) is significantly differentfrom the median (0.466) and mode (0.461). Note that the deviationsinduced by bubbles are both positive and negative-going. Theassymetrical skewing of the signal due to bubbling generally increasesas the yeast concentration decreases. For example, FIGS. 4e-h , show thesignals measured at 0.4 g/L yeast concentration under conditions of nobubbling (FIG. 4e-f ) and high bubbling (FIGS. 4g-h ). As shown in Table1, the mean signal increases by 77% as a result of the bubbling, whereasother measures of central value that are less dependent on outliers,such as the trimmed mean, median, and mode, are less dependent on spargerate. However, considered on their own (e.g. without also consideringstatistical measures of the distribution), none of the central valuestatistics tested so far are able to completely eliminate the effect ofbubbles under all conditions.

TABLE 1 Dependence of central signal value on bubbling rate Yeast SpargeAgitat. Conc. Rate Rate % Trimmed % % % File name (g/L) (Lpm) (rpm) Meanchange Mean change Median change Mode change 20130925003 200 0 400 4.7914.776 4.773 4.760 20130925030 200 16 900 5.680 19% 5.943  24% 6.006  26%6.142 29% 20130925093 25 0 400 0.462 0.460 0.460 0.460 20130925120 25 16900 0.508 10% 0.483 5.0% 0.480 4.3% 0.470 2.2%  20130927033 3.2 0 4000.0940 0.0932 0.0932 0.0927 20130927059 3.2 16 900 0.117 24% 0.0937 0.5%0.0932 0.0% 0.0927 0.0%  20130923393 0.4 0 400 0.0168 0.0159 0.01580.0150 20130923420 0.4 16 900 0.0297 77% 0.0173 8.8% 0.0172 8.9% 0.016913% 20130923303 0.05 0 400 0.00395 0.00345 0.00342 0.00306 201309233300.05 16 900 0.0337 750%  0.00561  63% 0.00521  52% 0.00366 20%20130923213 0.0064 0 400 0.00163 0.00132 0.00129 0.00107 201309232400.0064 16 900 0.0382 2200%  0.00441 230%  0.00376 191%  0.00178 66%

EXAMPLE 4 Mitigation of Bubbling Effects

Using the data set as described in Example 3, several differentstatistical estimates of the central value and distribution werecomputed, and their efficacy at mitigating the effects of bubbling wereevaluated. The central value of each data set was estimated by 4methods: (1) mean, (2) median, (3) mode, and (4) trimmed mean. Thetrimmed mean was computed by first sorting the data, then discarding theupper and lower quartile of the data, and computing the mean of theremaining points. The number of bins in the histogram used to computethe mode was generated by dividing the full range by the interquartilerange, multiplying by a factor between 3 and 30, and adding a termbetween 5 and 25. The distribution of each set was estimated by 6methods: (1) range, (2) standard deviation, (3) interquartile range, (4)mean absolute deviation, (5) median absolute deviation, and (6) modeabsolute deviation. The interquartile range was computed by sorting thedata and then computing the difference between the values of the datapoints at the 25th and 75th percentiles.

It is apparent from FIGS. 4a -h that bubbling has a dramatic effect onthe signal distribution. Measurement of the signal distribution incombination with measurement of the central value, was found to providean effective means of diminishing or eliminating the effect of bubbleson the reported biomass. FIG. 5a shows a plot of the Standard Deviationversus the Mean determined using the S1-D4 pair (1310 nm source, S-Dseparation 6.7 mm). Each yeast level in the figure is represented with adifferent symbol and color; the displayed yeast range was limited to 0.1to 25 g/L. The multiple points at each yeast concentration representmeasurements collected under different sparge and agitation conditions,with the data points collected under the highest bubbling and agitationrates generally appearing at the upper right of the figure. At eachyeast concentration a line was fit to the Mean as a function of theStandard Deviation. The fit was performed in log-log space. The percentRMS difference between the fit and the actual data, computed across allof the yeast concentration was 59%. Several limitations of thismeasurement configuration are evident from this plot:

-   i. In the absence of bubbles, the low range biomass limit is about    0.1 g/L.-   ii. If only the mean is used to compute the biomass, the presence of    bubbles can make it difficult to resolve biomass below about 3 g/L.-   iii. The linear range of response to biomass is limited to about 10    g/L. Between 25 and 50 g/L the reflectance signal rolls over and    becomes non-monotonic.-   iv. Even at the highest linear biomass (-10 g/L), bubbles can have a    significant effect on the reported biomass accuracy.-   v. Using the standard deviation in combination with the mean to    cluster the different yeast concentrations is only effective above    about 1.6 g/L.

The beneficial effect of decreasing the source-detector separation canbe seen by comparing FIGS. 5a -d in which the respective S-D separationsare about 7.1, 3.4, 1.2, and 0.22 mm. At the smallest S-D separation(0.22 mm, FIG. 5d ) the resolvable biomass range is extended at both thelow and high ends. Further improvement in the data clustering withineach yeast concentration results from the use of statistics that aremore robust to outliers.

Tables 2a and 2b show a comparision of the net RMS fitting errorsresulting from linear fits to a central value measure versus a measureof the distribution. The best results were observed when using eitherthe median or the trimmed mean as the central value measure, and usingeither the interquartile range or median absolute deviation as thedistribution measure. Plots of the median absolute deviation versus themedian are shown if FIGS. 5e and 5f , for the S1-D1 (1310 nm) and S2-D1(1550 nm) source-detector pairs, respectively. Compared to the plot ofstandard deviation vs mean (FIG. 5d ), the plot of mean absolutedeviation vs median (FIG. 5e ) for S1-D1 shows reduced deviation fromlinearity and better separation between biomass clusters, particularlyin the low biomass range, so that the resolvable biomass range isextended by about a factor of 10.

TABLE 2a S1-D1: Comparison of fitting errors for different statisticalmeasurements of central value and distribution. Mean Median Mode TrimmedMean Range 32% 9.3% 5.8%  11% Interquartile Range 26% 2.8% 3.3% 2.7%Standard Deviation 12% 8.6% 5.2% 9.8% Mean Abs. Dev. 6.3%  8.0% 4.9%9.1% Median Abs. Dev. 27% 2.6% 3.2% 2.6% Mode Abs. Dev. 33% 3.3% 5.7%3.5%

TABLE 2b S2-D1: Comparison of fitting errors for different statisticalmeasurements of central value and distribution. Mean Median Mode TrimmedMean Range 61% 6.1% 13% 6.5% Interquartile Range 54% 3.1% 11% 3.0%Standard Deviation 26% 4.8% 12% 5.1% Mean Abs. Dev. 9.2%  4.3% 12% 4.5%Median Abs. Dev. 53% 2.9% 11% 2.7% Mode Abs. Dev. 46% 3.4% 12% 3.4%

EXAMPLE 5 Bandwidth and Data Processing Requirements

Using the same data set as in Examples 3 and 4, frequency analysis wasperformed and additional data pre-processing methods were applied toevaluate the necessary bandwidth and processing power to achieve highdiscrimination against bubbles. Fourier Transform power spectra areshown in FIG. 6a-c , under conditions of high biomass and high sparge (6a), medium biomass and high sparge (6 b), and medium biomass and nosparge (6 c). FIG. 6a shows that at high biomass the majority of thebubbles are resolved at frequencies below about 400 Hz (800 Hzbandwidth). At medium biomass, but with the same agitation and spargeconditions (FIG. 6b ), the power spectrum is less steeply sloped, andthe majority of bubbles are resolved at frequencies below about 300-350Hz (600-700 Hz band width). With sparge turned off at medium biomass(FIG. 6c ), the power spectrum is nearly flat.

Additional variable data processing steps included: (1) averaging of alldata points within each laser pulse, (2) decimating the data by factorsof 2-8, and (3) truncating the data sets by factors of 2-16. Decimationwas performed by averaging D adjacent data points, where the decimationfactor is D. Truncation was performed by dividing the number of datasamples, N, by the truncation factor M, and keeping only the first N/Msamples in the data set. Decimation and truncation were always performedafter averaging of the data points within each laser pulse. When bothdecimation and truncation were performed on the same data set,decimation was performed first.

The minimum necessary bandwidth and number of data points were exploredby decimating and/or truncating the data sets and then comparing thelinear fitting errors for plots of the median absolute deviation versusthe median, across a wide range of yeast concentrations. The results aresummarized in Tables 3a and 3b, respectively, for the S1-D1 (1310 nm)and and S2-D1 (1550 nm) source-detector pairs. FIGS. 7a and 7b show thefitting results for the S1-D1 and S2-D1 source-detector pairs,respectively, with the decimation factor set to 2 (500 Hz band width)and the truncation factor set to 4 (1250 data points). Compared to theresults with no within-pulse averaging, at full bandwith (1 kHz) and notruncation (2×105 data points) (FIGS. 5e and 5f ), the decimation andtruncation have little apparent effect on the ability to accuratelyresolve biomass in the presence of bubbles. The dependence of the NetRMS Error on the truncation factor is relatively weak, and can beincreased up to 64 while maintaining the Net RMS Error below 6%. FIGS.7a and 7b respectively show the S1-D1 and S2-D1 fitting results fordecimation factor 2 and truncation factor 64 (N=78). Loss of resolutionis most evident in the low biomass range (compared to FIGS. 6e and 6f ).

TABLE 3a S1-D1: Comparison of fitting errors (median absolute deviationvs. median) after different levels of data decimation and truncation.Net Resolveable Decimation Truncation Bandwidth Number of RMS YeastRange Factor (D) Factor (M) (Hz) Points (N) Error (g/L) 1 1 1000 10,0002.77% 0.01-200 2 1 500 5,000 3.11% 0.01-200 4 1 250 2,500 3.86% 0.02-2008 1 125 1,250 5.16% 0.05-200 1 2 1000 5,000 2.80% 0.01-200 1 4 10002,500 2.89% 0.01-200 1 8 1000 1,250 3.07% 0.01-200 1 16 1000 625 3.70%0.01-200 1 32 1000 312 4.19% 0.01-200 1 64 1000 156 4.69% 0.02-200 1 1281000 78 12.2% 0.05-200 2 2 500 2,500 3.15% 0.01-200 2 4 500 1,250 3.17%0.01-200 2 8 500 625 3.23% 0.01-200 2 16 500 312 4.04% 0.02-200 2 32 500156 4.71% 0.02-200 2 64 500 78 5.11% 0.02-200 2 128 500 39 12.2%0.05-200 4 16 250 156 4.78% 0.02-200 4 32 250 78 5.65% 0.02-200 4 64 25039 5.96% 0.05-200 4 128 250 19 13.7%  0.1-200

TABLE 3b S2-D1: Comparison of fitting errors (median absolute deviationvs. median) after different levels of data decimation and truncation.Net Resolveable Decimation Truncation Bandwidth Number of RMS YeastRange Factor (D) Factor (M) (Hz) Points (N) Error (g/L) 1 1 1000 10,0003.09% 0.02-200 2 1 500 5,000 3.04% 0.02-200 4 1 250 2,500 3.03% 0.02-2008 1 125 1,250 3.33% 0.02-200 1 2 1000 5,000 3.08% 0.02-200 1 4 10002,500 3.17% 0.02-200 1 8 1000 1,250 3.61% 0.02-200 1 16 1000 625 3.88%0.05-200 1 32 1000 312 4.68% 0.05-200 1 64 1000 156 5.65% 0.05-200 1 1281000 78 9.12% 0.05-200 2 2 500 2,500 3.21% 0.02-200 2 4 500 1,250 3.12%0.02-200 2 8 500 625 3.61% 0.02-200 2 16 500 312 3.67% 0.02-200 2 32 500156 4.71% 0.02-200 2 64 500 78 5.55% 0.05-200 2 128 500 39 9.43%0.05-200 4 16 250 156 3.88% 0.02-200 4 32 250 78 5.19% 0.05-200 4 64 25039 6.56% 0.05-200 4 128 250 19 10.5% 0.05-200

Processing of the data in 2 steps could reduce the processing power andmemory requirements of the apparatus. 2-stage methods for computing thecentral value and distribution are compared in Tables 4 and 5. In thefirst data processing stage, the central value was estimated by 3methods (trimmed mean, median, and mode) and the distribution wasestimated by 3 methods (range, interquartile “IQ” range, and medianabsolute deviation “MAD”). In the second data processing stage, each ofthe 3 central value methods was applied to each of the first stageestimates, resulting in 9 combinations of methods (the second stagemethod is shown in parentheses in the row and column labels). Thedistributions were computed in the second stage by applying the threecentral value estimation methods to the distributions computed in thefirst stage.

The results shown in Tables 4a and 4b were determined with the number ofdata points fixed at 32 and the trim percentages fixed at 25% in bothstages. The lowest fitting errors resulted when the MAD method was usedto estimate the distribution in the first data processing stage. Allmethods using the mode to estimate the central value gave significantlyhigher fitting errors compared to those employing the trimmed mean ormedian.

TABLE 4a S1-D1: Comparison of 2 step data processing methods (N1 = 32,N2 = 32, Trim1 = 25%, Trim2 = 25%, 0.01-200 g/L, 400-900 rpm, 0-16 lpm).Tr. Mean Tr. Mean Tr. Mean Median Median Median Mode Mode Mode (Tr.Mean) (Median) (Mode) (Tr. Mean) (Median) (Mode) (Tr. Mean) (Median)(Mode) Range 7.29 7.11 11.2 6.49 6.14 9.97 7.61 8.72 18.8 (Tr. Mean)Range 6.71 6.55 10.6 5.97 5.65 9.73 8.20 7.76 18.8 (Median) Range 9.078.74 11.4 7.98 7.52 10.0 11.1 11.0 18.6 (Mode) IQ Range 3.64 3.64 9.953.69 3.57 8.96 8.45 7.22 16.9 (Tr. Mean) IQ Range 3.80 3.62 10.0 3.803.62 9.11 9.11 7.53 16.7 (Median) IQ Range 11.9 11.4 10.5 10.4 9.85 12.217.4 15.7 19.5 (Mode) MAD 3.33 3.45 9.63 3.31 3.30 8.77 8.67 7.18 17.4(Tr. Mean) MAD 3.40 3.38 9.59 3.34 3.26 8.70 8.76 7.18 17.5 (Median) MAD11.1 10.6 8.04 9.62 9.06 6.86 17.4 15.1 19.9 (Mode)

TABLE 4b S2-D1: Comparison of 2 step data processing methods (N1 = 32,N2 = 32, Trim1 = 25%, Trim2 = 25%, 0.01-200 g/L, 400-900 rpm, 0-16 lpm).Tr. Mean Tr. Mean Tr. Mean Median Median Median Mode Mode Mode (Tr.Mean) (Median) (Mode) (Tr. Mean) (Median) (Mode) (Tr. Mean) (Median)(Mode) Range 5.16 4.33 14.6 4.52 4.12 9.56 7.52 6.82 31.1 (Tr. Mean)Range 5.32 4.44 14.6 4.64 4.22 9.52 9.02 6.51 32.1 (Median) Range 6.725.79 15.4 5.90 5.37 10.1 14.0 9.52 20.7 (Mode) IQ Range 3.76 4.43 14.43.94 4.14 9.56 10.6 8.57 30.5 (Tr. Mean) IQ Range 4.06 3.84 14.6 4.033.91 9.70 8.63 7.12 30.7 (Median) IQ Range 7.80 6.81 10.3 6.87 6.21 11.617.3 11.4 35.2 (Mode) MAD 3.76 4.27 14.4 3.80 3.99 9.61 10.6 8.44 30.1(Tr. Mean) MAD 4.14 3.88 14.6 3.91 3.83 9.76 9.72 7.84 30.2 (Median) MAD7.34 6.58 12.4 6.53 5.97 7.67 16.6 11.3 37.3 (Mode)

Three of the methods giving the best results in Tables 4a and 4b werefurther explored by varying the number of data points (N1, N2) and trimpercentages (Trim 1, Trim 2) in the two processing stages. The results(Tables 5a and 5b) show that the fitting is only weakly dependent on theratio of N1 to N2 and the trim percentages. A more significantdependence is seen on the total number of data point (N1+N2).

TABLE 5a S1-D1: Comparison of 2 step data processing methods (0.01-200g/L, 400-900 RPM, 0-16 LPM). Tr. Mean Med. Med. (Tr. Mean): (Tr. Mean):(Med.): MAD MAD MAD N1 Trim 1 N2 Trim 2 (Tr. Mean) (Tr. Mean) (Med.) 12825 8 25 3.37 3.42 3.47 64 25 16 25 3.48 3.48 3.73 32 25 32 25 3.33 3.313.26 16 25 64 25 3.11 3.13 3.23 8 25 128 25 2.93 2.84 3.05 32 35 32 353.25 3.22 3.26 32 15 32 15 3.50 3.49 3.26 32 15 32 35 3.43 3.22 3.26 3225 16 25 4.69 4.73 4.70 32 25 8 25 5.14 5.07 5.13 16 25 32 25 4.52 4.774.31 8 25 32 25 5.01 4.90 5.25 8 35 128 35 2.79 2.90 3.05

TABLE 5b S2-D1: Comparison of 2 step data processing methods (0.02-200g/L, 400-900 RPM, 0-16 LPM). Tr. Mean Med. Med. (Tr. Mean): (Tr. Mean):(Med.): MAD MAD MAD N1 Trim 1 N2 Trim 2 (Tr. Mean) (Tr. Mean) (Med.) 12825 8 25 3.64 3.67 3.82 64 25 16 25 3.76 3.78 4.05 32 25 32 25 3.76 3.803.83 16 25 64 25 3.82 3.80 3.99 8 25 128 25 4.06 4.10 4.29 32 35 32 353.79 3.84 3.83 32 15 32 15 4.24 3.80 3.83 32 15 32 35 3.89 3.84 3.83 3225 16 25 4.63 4.86 5.47 32 25 8 25 5.84 5.70 5.60 16 25 32 25 4.39 4.404.71 8 25 32 25 6.35 6.22 6.17 8 35 128 35 4.13 4.21 4.29

EXAMPLE 6 Extrapolation of Immersion Sensor Results to Non-InvasiveSensor Measurements

The fiber immersion sensor results indicate that the widest biomassrange and best biomass clustering across different bubbling conditionsare obtained with the Source-Detector separation minimized (e.g.,close-packed optical fibers). For non-invasive measurements, anintervening layer of glass or plastic (the container wall) will separatethe sensor from the medium. The effect of this intervening layer on thesource-detector overlap was modeled, with the approximation that themean optical depth corresponds to the point of overlap of rays withmaximum divergence from and to the center axis of the source anddetector fibers, respectively.

The optical model is depicted in FIG. 8, and the results are summarizedin Table 6. Configuration 1 in Table 6 corresponds to an immersionsensor, for which the source (D1) and detector (D2) fibers both have 0.2mm core diameters and numerical apertures of 0.22. For thisconfiguration, the wall thickness, T, was set to 0, and the edge-to-edgeseparation between the source and detector fiber cores, Y was set to0.04 mm (closest-packing allowed for a fiber cladding diameter of 0.22mm). The resulting source-detector overlap depth, X, within the medium,is 0.71 mm. All other configurations in Table 6 correspond tonon-invasive measurements through a wall with thickness, T. For thenon-invasive configurations, the overlap depth, X, was fixed at 0.71,and the resultant edge-to-edge separation between the fiber cores, Y,was determined. The closest point of intersection between the source anddetector fiber cones, Z, within the container wall was also determined.A negative value of Z indicates that the source and detector cones willoverlap within the container wall, with the result that back (specular)reflections could be observed from the wall-medium interface. In thecase of configurations 2-10, the wall thickness is 2.1 mm. As shown forconfiguration 3 in Table 6, if the fiber size and type is kept the sameas in the immersion sensor case (configuration 1), but the optical conestravel through an intervening glass wall that is 2.1 mm thickness, anedge-to-edge fiber core separation, Y=0.66 mm, will result in the sameoverlap depth within the medium as in the immersion sensor case.

Y scales linearly with the wall thickness, so for configuration 11having a 1 mm wall thickness, Y=0.34 mm, while for a typical lab-scaleglass fermentor (1-12 L volume) with 6 mm wall thickness (configuration12), Y=01.8. If a sensor with the fiber edge-to-edge separationoptimized for a vessel with 2.1 mm thickness (Configuration 3, Y=0.66mm) was used on a larger vessel with a 6 mm wall, Z would be negative,indicating that the sensor would pick up back-reflections from theinterface between the container wall and the medium. On the other hand,if a configuration optimized for a 2.1 mm wall thickness (Configuration3) was used on vessel with a 1 mm thick wall (Configuration 11), the lowand high biomass performance of the sensor would be compromised, due tothe decreased overlap between the source and detector fibers.

The effects of varying the fiber diameter and numerical aperture aremodeled in cases 2, 4-10, and 14-16 of Table 6. In general, use ofsmaller diameter or numerical aperture fiber decreases Y, but withattendant loss of light collection efficiency of the detection fiber.For devices employing a light source with small diameter and numericalaperture, such as many laser sources, the use of small diametermulti-mode or single mode fiber provides high light delivery efficiency,while allowing Y to be decreased. Therefore, in some embodiments of thepresent invention optimal performance is attained by the use of a lasersource coupled to single mode optical fiber and one or more detectorscoupled to multi-mode optical fibers.

TABLE 6 Source-Detector Overlap Calculations. Inputs ResultsConfiguration D₁ D₂ T X Y Z # (mm) (mm) (mm) NA₁ NA₂ n_(g) n_(a) (mm)(mm) (mm) 1 0.2 0.2 0 0.22 0.22 1.5 1.33 0.715 0.040 0.04 2 0.1 0.1 2.10.22 0.22 1.5 1.33 0.715 0.763 0.14 3 0.2 0.2 2.1 0.22 0.22 1.5 1.330.715 0.663 0.04 4 0.4 0.4 2.1 0.22 0.22 1.5 1.33 0.715 0.463 −0.16 50.6 0.6 2.1 0.22 0.22 1.5 1.33 0.715 0.263 −0.36 6 0.1 0.2 2.1 0.22 0.221.5 1.33 0.715 0.713 0.09 7 0.1 0.2 2.1 0.22 0.39 1.5 1.33 0.715 1.0660.19 8 0.1 0.2 2.1 0.22 0.48 1.5 1.33 0.715 1.268 0.25 9 0.1 0.2 2.10.39 0.39 1.5 1.33 0.715 1.420 0.29 10 0.1 0.2 2.1 0.48 0.48 1.5 1.330.715 1.822 0.40 11 0.2 0.2 1 0.22 0.22 1.5 1.33 0.715 0.337 0.04 12 0.20.2 6 0.22 0.22 1.5 1.33 0.715 1.819 0.04 13 0.2 0.2 12 0.22 0.22 1.51.33 0.715 3.598 0.04 14 0.1 0.2 1 0.22 0.39 1.5 1.33 0.715 0.607 0.1915 0.1 0.2 1 0.39 0.39 1.5 1.33 0.715 0.827 0.29 16 0.1 0.2 1 0.48 0.481.5 1.33 0.715 1.079 0.40 X: Depth at which center cones of the sourceand detector fibers overlap within the medium. Y: Edge-to-edgeseparation between the source and detector fiber cores. Z: Closest pointof intersection between the source and detector fiber cones within thecontainer wall.

EXAMPLE 7 Non-Invasive Fiber Optic Sensor for a Plastic Vessel

A fiber optic ferrule, as depicted in FIG. 14, was designed andconstructed for making measurements through the bottom of a 1 mm thickclear plastic vessel. Vessels constructed from both Makrolon and Luranmaterials were tested. Source light was provided by a 1330 nm, 30 mWdiode laser (Applied Optoelectronics, part number 01-05-1182DFB-1330-BF-25-CW-SA-N182) with a songle mode fiber output that wassplit into 16 equal channels by a single mode fiber optic splitter(Neptec OS, Inc., part number SCSMDWCM). The light source penetrationdepth at this wavelength is limited to about 25 mm by water absorbance,allowing accurate reflectance measurements at low biomass even inshallow fluid depths. The sixteen fiber channels provide simultaneouslight source excitation for up to sixteen fiber optic ferrules. Thelight source fibers (both in the splitter and in the ferrule) weresingle mode fibers (Corning Optical Fiber, part number SMF28e), whilethe detection fibers were 200 μm core multi-mode fibers (ThorLabs, partnumber FG200LC). The use of single mode optical fiber for source lightconveyance has the benefit of lower cost for the fiber optic splittercompared to a multi-mode fiber splitter, as well as reducing the amountof specular reflectance from the vessel for a given source-detectorseparation and window thickness, compared to multi-mode fiber. The useof multi-mode optical fiber for detection allows capture of thediffusely reflected source light over a relatively large surface area,resulting in much higher collection efficiency compared to that providedby single mode fiber.

The detection fibers were individually detected, amplified, andmultiplexed into a single analog-to-digital converter (ADC), as furtherdescribed in Example 11. The multiplexing helps to reduce the overallsystem cost for monitoring multiple vessels simultaneously. The laserwas modulated at approximately 781 Hz with a 75% duty cycle. The ADCoutput consisted of 16-bit integers streaming at a rate of 50 kHz. AProportion-Integral-Derivative (PID) control loop was used to maintainthe laser output power at the desired level by using the signal from abuilt-in laser monitoring diode as a feedback signal read by themicroprocessor. Each laser modulation cycle consisted of 64 detectorsamples: 16 samples with the laser off followed by 48 samples with thelaser on. Each measurement consisted of 2040 laser pulses (serieslasting about 2.6 seconds). The first three points in the “laser off'and “laser on” periods were discarded and all remaining points wereaveraged and the resultant laser off signal was subtracted from thelaser on signal to provide a demodulated reflectance signal. Table 7summarizes the scheme for accomplishing this automatic sensitivityranging, where the Analog-to-Digital Converter (ADC) count thresholdsshown in the first column are user-controlled variables stored ininstrument memory. If the ADC count is above a third threshold value, T3(=40,000 in Table 7), the detector gain is decreased if it is notalready at the minimum value. If the detector gain is already at theminimum value then the laser power is decreased by a factor, f In thisexample f was selected to be 2. This process is repeated until the ADCcounts fall below T3. On the other end of the scale, if the ADC countsare below a first threshold value, T1 (300 in Table 2), the laser poweris increased if it is not already at a maximum allowed value. If thelaser power is already at the maximum allowed value then the detectorgain is increased. This process is repeated until the ADC counts areraised above Ti. If the ADC counts fall between T1 and a secondthreshold, T2 (16,000 in Table 7), then the detector gain is maintained,but the laser power is increased by factors of f, until the ADC countsexceed T2. In this example, the sensitivity ranging was only activatedwhen at least n sequential measurements indicated the need foradjustment, where n was set to 3. This helped prevent toggling of thesensitivity settings during noisy measurement periods. For optimalperformance n could be decreased or increased depending on the amount ofmeasurement noise. This scheme greatly increases the dynamic range ofthe instrument, as is necessary in many applications, such as biomassmeasurements ranging over many orders of magnitude of biomass. Themedian and median absolute deviation (MAD) of the demodulatedreflectance signal were computed in two stages, as described in Example5, above. The detector gain and laser power were automatically varied inorder to keep the signals within a desired range of amplitudes. Themedian and MAD signals were normalized by the detector gain and laserpower at which they were measured.

TABLE 7 Laser Power and Detector Gain Ranging Scheme ADC Counts LaserPower Detector Gain 65536 Decrease if detector gain is at Decrease ifnot at minimum. minimum. . . . Decrease if detector gain is at Decreaseif not at minimum. minimum. 40000 Decrease if detector gain is atDecrease if not at minimum. minimum. 39999 Maintain. Maintain. . . .Maintain. Maintain. 16000 Maintain. Maintain. 15999 Increase if not atmaximum. Maintain. . . . Increase if not at maximum. Maintain.  300Increase if not at maximum. Maintain.  299 Increase if not at maximum.Increase if laser is at maximum. . . . Increase if not at maximum.Increase if laser is at maximum.   0 Increase if not at maximum.Increase if laser is at maximum.

The “bubble calibration” shown in FIG. 15 was created by running 14yeast concentrations under a variety of agitation and sparge conditions.A fixture was constructed to hold the fiber optic ferrule against thebottom of a 250 mL vessel. The vessel contained an agitator with twoRushton impellers, a sparge tube, and a pH probe. The yeastconcentrations (shown in the legend of FIG. 15) ranged between 0.02 and200 g/L dry cell weight; the agitation rate was varied between 1000 and4500 rpm; and the room-air sparging ranged between 0 and 2 VVM (volumeof gas per volume of medium per minute). The markers shown in FIG. 15depict the measurements overlaid on which are linear fits.

This bubble calibration, consisting of the linear slopes, intercepts,and limits at each of the 14 yeast concentrations, was saved intoinstrument memory and then applied to subsequent measurements. Thelocation of each new measurement, consisting of a median and MADreflectance value, was determined on the bubble calibration map (FIG.15), and the nearest 2 calibration lines were interpolated between tocreate a calibration line for the new measurement. The minimumreflectance of this interpolated calibration line was then reported asthe “bubble-free” reflectance, R₀. FIG. 16 summarizes the testingresults before (median reflectance) and after (R₀) bubble correctionunder a variety of yeast concentrations, and agitation and spargeconditions. As depicted in the top left graph of FIG. 16, within each ofthe 7 tested yeast concentrations, the agitation and sparge conditionswere increased in 3 steps: (1) 1000 rpm, 0 VVM, (2) 2500 rpm, 1 VVM, and(3) 4000 rpm, 2 VVM. As can be seen by comparing the left and rightsides of FIG. 16, the agitation and sparge rate caused significantincreases in the measured median reflectance at all yeastconcentrations, but after “bubble correction” these effects were largelyremoved.

Bubble-compensation has the effect of improving the correlation betweenthe bubble-compensated reflectance and biomass. In many cases thisrelationship can be accurately characterized by a linear fit in log-logspace, such as shown in FIG. 17. These data were collected on a 15 mLLuran vessel filled with 12 mL of media. The linear relationship betweenbiomass and bubble-compensated reflectance holds over more than 3 ordersof magnitude of biomass concentration.

EXAMPLE 8 Immersible Probe with Symmetrical Detectors

Another example of accurate biomass conversion in the presence of strongpotential interference from bubbles was demonstrated using an immersiblefiber optic probe. Three 200/220 μm core/cladding optical fibers werelinearly close-packed, as depicted in FIG. 18. The center fiber wasselected for the source, so that the two surrounding detection fiberswere symmetrically positioned relative to the source. A 3 mW, 1310 nmdiode laser (Xiamen Bely Communication Equip. Co., part numberBLLD-RA-F3130B-1GR) was connected to the source fiber. The two detectorfibers were connected to 1 mm diameter InGaAs detectors (Xiamen BelyCommunication Equip. Co., part number BLPD-RA-1KAR-B). In someexperiments, a bandpass interference filter centered near 1310 nm wasadded in front of the detector element, to discriminate against ambientlight. Both the laser and detectors included fiber optic connectors,simplifying the coupling between the fibers and opto-electroniccomponents. Except for the fiber optic apertures, the stainless steelexterior of the probe was coated with titanium nitride (“TiN”). Thiscoating was found to reduce the adhesion of bubbles to the probesurface, as described in Example 12. The curvature of the sensor face(see “detail A” in FIG. 18) was also found to be effective atdiscouraging bubble adhesion. Comparison of the reflectance measured bythe two symmetrically-placed detectors was found to be an effectivemethod of eliminating measurements where interference was present. Forexample, by setting a threshold for agreement between the two detectors,measurements made during periods where one or more bubbles were presentat the sensor tip could be easily rejected.

The response of the two detectors was normalized to each other using aquadratic polynomial correction in log-log space to the medianreflectance measured by each detector over a wide range of biomass underconditions of no sparging and moderate agitation. This type ofcorrection was also found to be effective at compensating formanufacturing differences between different sensors. Changes in opticalcoupling efficiency over time could be corrected with a linearcorrection based on measurements of a material having fixed reflectanceproperties.

The probe was immersed in a 250 mL glass vessel containing 200 mL ofmedia, dual Rushton impellers, a sparge tube, metal baffles, and severalother probes (temperature, pH, dissolved oxygen). The tip of the probewas held at least 3 cm from the bottom of the vessel during allmeasurements. A bubble calibration was collected, following a similarprocess to that described in Example 7, except that additionalmeasurements were collected at low biomass (to 0.001 g/L dry cell weightof yeast). The bubble calibration was tested under conditions of low andhigh bubbling, with the result shown in FIG. 19. Under both bubblingconditions, the concentration prediction error was less than 5% overmore than 4 orders of magnitude of biomass (yeast dry cell weight0.01-200 g/L).

EXAMPLE 9 Applicability of Bubble Correction Across Different Organisms

The bubble calibration collected using yeast in Example 8 was appliedwithout modification to subsequent measurements on a culture ofescherichia coli. As can be seen in FIG. 20, the bubble-correction waseffective over at least 4 orders of magnitude of biomass (E. coli drycell weight), despite the large difference in the cell size and shape ofthe organism used to generate the bubble calibration (saccharomycescerevisiae) and that used to test the bubble calibration (e. coli). Thisresult demonstrates that the bubble correction method is not limited tothe particular organism used to generate the bubble calibration.

EXAMPLE 10 Effect of Dissolved Protein on Bubble Correction Mapping

Micro-organisms are frequently genetically engineered to express aparticular protein of interest. In order to test the effect of proteinexpression on bubble calibration, Albumin (chicken egg white, ScienceLab Supplies, part number C1210-30G) was added to yeast suspensions.Three yeast concentrations were tested: 0.2, 2, and 20 g/L dry cellweight. And at each yeast concentration, up to three levels of relativealbumin concentration were tested: 1%, 5%, and 10%. The addition ofalbumin to the media resulted in greatly increased bubble retentionunder some conditions. As shown in FIG. 21, this increased bubbleretention resulted in a non-linear shape to the MAD-Median mapping atthe highest albumin concentrations. In order to compensate for bubblesin the presence of high protein concentrations a non-linear mapping maytherefore be more effective than linear mapping. In some embodiments atleast a 2^(nd) order polynomial fit is used for the bubble calibration,thereby extending the calibration to compensate for the types of extremefoaming that can be observed in the presence of high proteinconcentrations.

EXAMPLE 11 Electrical Schematic for Single Sensor and MultiplexingSystems

A microprocessor can U6 provide& timing control of a laser and twodetectors. Digital laser control signals from the microprocessor areconverted into an analog signal (voltage), switched between high and lowstates by, and converted to currents to power the laser. A lasermonitoring diode is amplified provided as a feedback to themicroprocessor. The signals from the two detectors, are firsttrans-impedance amplified, and then further amplified with variable gainamplifiers. The gain level is controlled by digital communicationbetween the microprocessor and gain switches. The amplified detectorsignals are digitized by analog-to-digital converters. Themicroprocessor uses digital signals to compute median and MADreflectance results, and applies corrections, normalizations, andcalibrations, using stored coefficients. The coefficients that are mostrelevant to sensor performance are stored in a memory device within thesensor, e.g., a 1-wire EEPROM. The microprocessor brings the measuredreflectance signals into an optimal range by varying both the laserpower and the detector gain. Communication between the microprocessorand other devices (e.g. a personal computer), is provided via aUniversal Serial Bus (USB) interface within the microprocessor.Electrostatic discharge protection is provided.

A high-level schematic for a multiplexed embodiment of the presentinvention is depicted in FIG. 23. A single laser is split using a fiberoptic splitter, providing light for multiple sensors simultaneously. Thereflected source light captured by the detection fibers in the ferrulesis individually detected and amplified, and then routed through amultiplexer before analog-to-digital (ADC) conversion. This arrangementprovides a low-cost method for monitoring the biomass in multiplevessels simultaneously.

EXAMPLE 12 Comparison of Uncoated and Coated Probes

Several probes were constructed of identical design to that described inExample 8, except that the titanium nitride coating was omitted, so thatthe outer surface of the probe tip surrounding the optical fibers wasbare polished stainless steel instead of titanium nitride coatedstainless steel. Testing was performed by immersing both uncoated andcoated probes in the same plastic 250 mL bioreactor vessel filled withmedium. Five yeast concentrations (0, 0.1, 1, 10, and 100 g/L) weretested under conditions of fixed 2 vvm room air sparging and variableagitation (500, 1500, and 2500 rpm) using a Rushton type impeller. Laserdriving and data acquisition were as described in Example 8. Within eachlaser pulse a 25% truncated mean was computed from the data pointscollected during the period that the laser was on. The resultingmeasurements were sorted into histograms as shown in FIG. 24.Differences between the uncoated and coated probes were particularlyevident under conditions of high biomass and sparging but relatively lowagitation rates, such as shown in the bottom two graphs in FIG. 24.Under these conditions the signal distribution was substantially morenarrow for the coated probe relative to the uncoated probe. Theseresults demonstrate reduced susceptibility of the titanium nitridecoated probe to bubble adhesion, under certain conditions. A more narrowsignal distribution implies that the median value can be estimated withhigher accuracy, leading to more accurate prediction of biomass.

EXAMPLE 13 Envisioned Combinations of Device Elements

The typical device for measuring the concentration of particles in amedia includes, e.g., one or more light sources to interrogate themedia, one or more light detectors, a container holding media with oneor more particle types. The light sources can emit light at onefrequency, or in a range of frequencies, in a direction characterized bythe optical axis (e.g., center of an illumination cone). The detectorscan detect along an optical axis along which light is received into thedetector.

Functional combinations of particle concentration assay devices caninclude several combinations of elements. For example, e.g., devices caninclude one light source and one detector; one light source and twodetectors, two light sources and one detector, one light source andthree detectors, one light source and four detectors, two light sources(having different emission wavelengths) and two detectors. Any of theabove combinations can be used to analyze a media in a container(through a wall transparent to the light source wavelength(s)) or thedetector and/or light source can be immersed in the media forinterrogation and detection. Any of the above combinations can be usedin combination with media containing one type of particle (e.g., abiologic cell) or two or more types of particles (e.g., gas bubbles,macromoleucle aggregates, and/or relatively large inorganic particles).Any of the above combinations can be used in combination with alternatealignments of detector and/or light source central optical axes, such asparallel light/detector axes, converging light/detector axes, normalincident optical axis at the optical interface with media, and/or tiltedorientation of light axis and/or detector axis at the media opticalinterface.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be clear to one skilledin the art from a reading of this disclosure that various changes inform and detail can be made without departing from the true scope of theinvention. For example, many of the techniques and apparatus describedabove can be used in various combinations and permutations, all of whichcannot reasonably be recited individually in this document, but can beunderstood by one of skill in the art on review of this specification.

All publications, patents, patent applications, and/or other documentscited in this application are incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent, patent application, and/or other document wereindividually indicated to be incorporated by reference for all purposes.

1-86. (canceled)
 87. A method of determining the concentration in amedium of a first particle type in the presence of a second particletype that comprises the steps of: a) passing light originating from afirst light source into the medium; b) detecting a first light signal,wherein the signal is from light originating from the first light sourcethat was reflected from within the medium, wherein the detectionbandwidth and the measurement volume are configured to allow signalfluctuations due to the second particle type to be resolved, whereas thesignal due to the first particle type is substantially constant at agiven concentration; c) separating the signal fluctuations from theconstant signal to determine the concentration of the first particletype; whereby the concentration of the first particle type is determinedwith reduced error, which error is due to background reflectance signalsfrom the second particle type in the medium.
 88. The method of claim 87,wherein the detection bandwidth is greater than 10 Hz.
 89. The method ofclaim 87, wherein the measurement volume is less than 200 mL.
 90. Themethod of claim 87, wherein the first particle type is at least 100-foldsmaller in volume than the second particle type.
 91. The method of claim87, wherein the number of particles within the volume of medium beingmeasured is at least 100-fold greater for the first particle type thanfor the second particle type. 92-103. (canceled)
 104. The method ofclaim 87, wherein the medium is aqueous and the first particle type isselected from the group consisting of: micro-organisms, animal cells,plant cells, polymer particles, proteins, nanoparticles, a sol gel, anda virus.
 105. The method of claim 104, wherein the second particle typeis selected from the group consisting of: gas bubbles, feedstock, growthmedia, complex sugars, cellulosic material, pulverized biologicalmatter, partially digested biological matter, aggregated proteins,products expressed by the first particle type, lipid/fat micelles,flocculated cells, filamentous clusters of cells, aggregated cells, andprecipitated solutes.
 106. The method of claim 104, wherein the secondparticle type comprises more than one category of particles, thecategories consisting of: gas bubbles, feedstock, growth media, complexsugars, cellulosic material, pulverized biological matter, partiallydigested biological matter, aggregated proteins, products expressed bythe first particle type, lipid/fat micelles, flocculated cells,filamentous clusters of cells, aggregated cells, and precipitatedsolutes.
 107. The method of claim 87, wherein the measurement volume isrestricted by selecting a source wavelength that has limited meanpenetration depth into the medium.
 108. The method of claim 107, whereinthe medium is aqueous and the source wavelength is selected from withinthe range of 1100 to 1400 nm.
 109. The method of claim 107, wherein themean penetration depth into the medium by the source wavelength in thesubstantial absence of scattering particles is between 1 and 50 mm. 110.The method of claim 87, further comprising restricting the measurementvolume using optical fibers to convey the source light toward the mediumand the scattered light within the medium toward the detector.
 111. Themethod of claim 87, further comprising using a processor to compute afirst statistical measure that is related to a central value of thedetector signal; and using the processor to compute a second statisticalmeasure that is related to a distribution of the signal.
 112. The methodof claim 111, further comprising mapping the first and secondstatistical measures over varying concentrations of the first and secondparticle type to reduce a contribution of the second particle type lightreflections to the concentration determinations of the first particletype.
 113. The method of claim 87, wherein a sensor houses the firstlight source and a first light detector, and further comprisingpositioning the sensor to allow the particle concentration measurementsto be made through a wall of a container holding the medium.
 114. Themethod of claim 113, further comprising selecting a distance between acentral optical axis of the first light source and a central opticalaxis of the first light detector at an interface between the containerand medium to minimize sensitivity to specular reflections from thecontainer wall, while also maximizing the range of sensitivity tobiomass changes.
 115. The method of claim 87, further comprisingselecting the measurement volume to avoid or reduce sensitivity tointerference due to specular reflectance from nearby objects in themedium, which objects are selected from the group consisting of:agitators, probes, sparge tubes, baffles, and a container wall.
 116. Adevice for determining the concentration in a medium of a first particletype in the presence of a second particle type, which device comprises:a sensor comprising a light source positioned to emit light into themedium; the sensor further comprising a detector positioned to detect asignal of the source light scattered by the particles within the medium;wherein a signal detection bandwidth and a measurement volume areselected to allow signal fluctuations due to the second particle type tobe resolved, whereas the signal contribution from the first particletype is substantially constant at a given concentration; an analogfilter or digital filter configured to substantially separate the signalfluctuations from the constant signal; a processor configured tocorrelate the filtered signals to the concentration of the firstparticle type; whereby the concentration of the first particle type isdetermined with reduced error, which error is due to reflectance signalsfrom the second particle type.
 117. The device of claim 116, wherein themedium is aqueous and the first particle type is a micro-organism,animal cell, or plant cell; wherein the second particle type comprisesone or more particle types selected from the group consisting of: gasbubbles, feedstock, growth media, complex sugars, cellulosic material,pulverized biological matter, partially digested biological matter,aggregated proteins, products expressed by the first particle type,lipid/fat micelles, flocculated cells, filamentous clusters of cells,aggregated cells, and precipitated solutes; whereby the concentration ofmicro-organisms or cells is determined over a wide range ofconcentrations, with reduced error from changing process conditions thatcause the concentration of the second particle type to vary.
 118. Thedevice of claim 116, wherein the medium is aqueous and the light sourcewavelength is selected to be in a spectral region where the light issubstantially absorbed by water, thereby limiting the measurementvolume.